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The genetic control of self-incompatibility
in sweet and sour cheny

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THE GENETIC CONTROL OF SELF-INCOMPATIBHJTY
IN SWEET AND SOUR CHERRY

By

Nathanael R. Hauck

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Plant Breeding & Genetics Program;
Department of Horticulture

2004

ABSTRACT

THE GENETIC CONTROL OF SELF-INCOMPATIBILITY
IN SWEET AND SOUR CHERRY

By

Nathanael R. Hauck

Gametophytic self-incompatibility (GSI) is a common mechanism for preventing
inbreeding in flowering plants. Typically, GSI in diploid species breaks down due to
polyploidy resulting in self-compatible (SC) tetraploid species. The diploid sweet cherry
and the tetraploid sour cherry represent an exception, as sour cherry individuals can be
either self-incompatible (SI) or SC. SI is undesirable for cultivation due to the
inefficiencies of growing pollinator varieties and the reliance on bees to ensure adequate
cross-pollination. Therefore, sour cherry breeders should develop SC selections.
Without an understanding of the genetic control of SI and SC in sour cherry, breeders are
not able to predict the 81 or SC phenotype of seedlings prior to the production of flowers,
which typically occurs 3-5 years after planting. The availability of molecular markers to
predict the SI or SC phenotype of a seedling could save valuable field space and
evaluation time. The goal of this dissertation was to determine the genetic control of SI
and SC in sour cherry. To do this, it was first necessary to determine which S-haplotypes
exist in sweet cherry, one of the progenitors of sour cherry. RFLP analyses were used to
determine the banding profiles for 14 sweet cherry S-haplotypes. Sour cherry was then
found to contain six of these sweet cherry S-haplotypes (S 1, S4, S6, S9, S12 and S13) in
addition to six unique S-haplotypes (S66, S6,", S0, Sb, S4 and Se). Using inter-specific

crosses between sweet and sour cherry and self-pollinations of sour cherry, four of the six

shared S-haplotypes (S 1, S4, S6, and S9) and one of the unique S—haplotypes (Sb) were
shown to be firnctional and capable of accomplishing S-haplotype-specific rejection of
pollen. The other S-haplotypes in sour cherry (S13, 56c, S6,”, S0, Sd, and Se) were shown to
be non-functional and incapable of initiating S-haplotype-specific rejection of pollen.
Finally, a hypothesis regarding the genetic control of SI and SC in sour cherry was
developed through the analysis of S-haplotype segregation in 794 progeny from six sour
cherry self-populations and 15 inter-specific crosses between sweet and sour cherry. SI
and SC predictions were verified using additional self-pollination and crossing
experiments. The data suggests that the partial breakdown of SI in sour cherry is due to
the accumulation of non-functional S-haplotypes that are incapable of S-haplotype-
specific rejection of pollen, rather than due to the competition between pollen-S products
in heteroallelic pollen, which is commonly observed in the Solanaceae. The implications

of these findings on sour cherry breeding and on our knowledge of GSI are discussed.

ACKNOWLEDGMENTS

I would like to send a special thanks to my committee members, Amy Iezzoni,
Jim Hancock, Mitch McGrath, Steve van Nocker, and Pam Green, for their guidance and
valuable suggestions throughout the years. No doubt, these suggestions have greatly
improved the quality of my research and dissertation. Amy has provided me with a
wonderfirl research project, has let me help in the writing or review of numerous
manuscripts and grant proposals, has introduced me to the value of collaboration and has
been a tremendous mentor, overall. Jim’s humor and ability to make me think deeply
about plant evolution not only made my stay at MSU more enjoyable, but also inspired
me to have a great interest in polyploid genome evolution. The numerous suggestions
from Mitch and Steve, both in and out of committee meetings, have been invaluable.

I would also like to thank most of the past and current members of the Iezzoni and
Hancock labs, especially Audrey Sebolt, Jim Olmstead, Chris Owens, Renate Karle, Pete
Callow, Cholani Weebadde and Chrislyn Drake. Each of them has made my stay at MSU
more interesting and contributed to the success of my project.

This project would also not be possible if not for the help of Ryutaro Tao from
Kyoto University, and two of his students Hisayo Yamane and Kazuo Ikeda. They have
taught me a lot about science and the Japanese culture.

In addition, I must thank Barb Sears and Jim Hancock for giving me the
opportunity to participate in their undergraduate genetics class and for helping me evolve

as a teacher. In addition, Barb’s advice and feedback over the past three years while

iv

serving as my teaching mentor has been invaluable. The numerous discussions about
teaching with Barb and Jim, as well as with Cholani Weebadde and Veronica Vallejo,
have been helpful.

I appreciate all of the support of my parents who instilled in me a desire to learn
and, somehow, an interest in plant genetics. A special thanks to my wife, Paula, who has

always supported and helped me when necessary.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii
LIST OF FIGURES .................................................................................. x
CHAPTER 1: LITERATURE REVIEW .......................................................... 1
INTRODUCTION ............................................................................... 2
LITERATURE REVIEW ....................................................................... 3
The Origin of Sour cherry ..................................................................... 4
The Genomic Structure of Cherry ............................................................ 4
Prevalence of Self-Incompatibility .......................................................... 5
Gametophytic Self-Incompatibility in Sweet Cherry ...................................... 6

The Mechanism of the RNase-Based Gametophytic Self-Incompatibility System....6
Proposed Mechanisms for the Breakdown of Gametophytic SI .......................... 9
REFERENCES ................................................................................... 13

CHAPTER 2: REVISITING THE S—ALLELE NOMENCLATURE IN SWEET

CHERRY (PRUN US A WUM) USING RFLP PROFILES .................. 19
ABSTRACT ...................................................................................... 20
INTRODUCTION ............................................................................... 21
MATERIALS AND METHODS .............................................................. 23
Plant Material ................................................................................ 23
DNA Isolation ................................................................................ 24
Genomic DNA Blot Analysis ............................................................. 24
PCR-amplification of S-alleles ......................................................... 24
RESULTS AND DISCUSSION ............................................................... 25
REFERENCES ................................................................................... 41

CHAPTER 3: SELF-COMPATIBILITY AND INCOMPATIBILITY IN TETRAPLOID
SOUR CHERRY (PRUNUS CERASUS L.) .................................. 44
ABSTRACT ...................................................................................... 45
INTRODUCTION ............................................................................... 46
MATERIALS AND METHODS .............................................................. 49
Plant Material ................................................................................ 49
Analysis of Pollen Tubes Grth ......................................................... 50
cDNA Cloning of S-RNases ............................................................... 51

DNA isolation and Southern analyses from the parents and progeny in the linkage
mapping population ................................................................... 52
Inheritance and linkage analysis .......................................................... 53
RESULTS ........................................................................................ 54

Pollen tube growth studies of sour cherry and sweet cherry interspecific crosses.54

vi

cDNA cloning from ‘RS’ .................................................................. 54
Inheritance and linkage analysis of the S-RNases in the ‘RS’ x ‘EB’ mapping

population ............................................................................... 56

SI and SC phenotypes of progeny from the ‘RS’ x ‘EB’ mapping population... . .63
DISCUSSION .................................................................................... 63
REFERENCES .................................................................................. 68

CHAPTER 4: GENETIC CONTROL OF SELF-INCOMPATIBILITY AND SELF-
COMPATIBILITY IN TETRAPLOII) SOUR CHERRY (PRUNUS

CERASUS L.) ..................................................................... 73
ABSTRACT ...................................................................................... 74
INTRODUCTION ............................................................................... 75
MATERIALS AND METHODS .............................................................. 78

Plant material ................................................................................ 78
Field crosses and self-pollinations ........................................................ 80
DNA isolation ............................................................................... 80
PCR amplification of S-RNases ........................................................... 83
S-genotype or S-phenotype determination for hypothesis testing ..................... 83
Analysis of pollen tube growth for hypothesis testing ................................. 85
RESULTS ........................................................................................ 86
Inter-specific crosses ........................................................................ 86
Sour cherry self-populations ............................................................... 99
Hypothesis verification .................................................................... 102
DISCUSSION ................................................................................... 105
REFERENCES ................................................................................. 116
CHAPTER 5: CONCLUSIONS, GENERAL DISCUSSION AND FUTURE

RESEARCH ....................................................................... 120

CONCLUSIONS AND GENERAL DISCUSSION ....................................... 121
Impact on sour cherry breeding ........................................................... 123
Implications for understanding the effect of polyploidy on GSI ..................... 125

FUTURE RESEARCH ........................................................................ 128

REFERENCES .................................................................................. 131

APPENDIX A: DNA EXTRACTION PROTOCOL FOR CHERRY SEED .............. 134

APPENDIX B: COLLECTION OF SELF-INCOMPATIBILITY DATA AND

SCHEMATIC REPRESENTATIONS OF OB SERVED S-HAPLOTYPE
SEGREGATION DATA ...................................................... 136

vii

Table 2.1:

Table 2.2:

Table 2.3:

Table 3.1:

Table 3.2:

Table 3.3:

Table 4.1:

Table 4.2:

Table 4.3:

Table 4.4:

Table 4.5:

LIST OF TABLES

Sizes of DNA restriction fragments for sweet cherry S—alleles used in this
study ..................................................................................... 29
S—allele genotypes of 17 sweet cherry cultivars used in this study .............. 30
S-genotypes for five Washington State University and Michigan sweet cherry

selections ................................................................................ 37

Cross-compatibility results for pollination of sour cherry styles with sweet
cherry pollen based on examination of pollen tube grth in styles 72 hours
after pollination ........................................................................ 55

Cross-compatibility results for pollination of sweet cherry cultivars with sour
cherry pollen based on examination of pollen tube growth in styles 72 hours

afier pollination ........................................................................ 55
RFLP segregation of S-RNase alleles in the ‘RS’ x ‘EB’ mapping

population .............................................................................. 61
Sweet and sour cherry cultivars, with their proposed S-haplotypes, used in this
study ..................................................................................... 79

Reciprocal inter-specific crosses between sour cherry and sweet cherry used
to investigate the functionality of the S4, S6, and Sg-haplotypes from sour

cherry .............................................................................................................. 81
Sour cherry self-populations analyzed to determine the functionality of the
513', Sq, Sb, 5.; and Se-haplotypes ..................................................... 82

DNA sequences, annealing temperature, and references for S-RNase
genotyping PCR primers used in this study ........................................ 84

Segregation of S-haplotypes and S-genotypes/phenotypes in triploid progeny

from sweet cherry x sour cherry reciprocal crosses to test the functionality of
the S4, S6 and Sg-hapltoypes in sour cherry ......................................... 87

viii

Table 4.6: Sour cherry self-pollinations to test the functionality of S 13 ', Sa, Sb, Sc, Sd and
Se ....................................................................................... 100

Table 4.7: The S-genotype, SI or SC predictions based on the S-genotype, and the SI or
SC phenotype of 92 sour cherry selections used to test the validity of the
hypothesis for the genetic control of SI and SC in sour cherry ................ 106

Table B. 1: The self-pollination of 60 sour cherry selections to test the validity of the
hypothesis for the genetic control of SI and SC in sour cherry. Predictions
were based on the knowledge of the S-genotypes of each tree ................. 137

Table B.2: Thirteen sour cherry crosses used to test the validity of the hypothesis for the
genetic control of SI and SC in sour cherry. The compatibility or
incompatibility of each cross was predicted based on the S-genotype of the
parents .................................................................................. 140

Table B3: The S-genotype and SI or SC phenotype for 81 progeny in the Rheinische
Schattenmorelle (RS) x Erdi B6term6 (EB) population ......................... 141

ix

LIST OF FIGURES

Figure 2.1: Genomic DNA blot analysis of 22 sweet cherry cultivars ....................... 26
Figure 2.2: PCR analysis of S-alleles from sweet cherry ....................................... 32

Figure 3.1 Amino acid sequence alignment of four S-RNases from ‘RS’, Sc, Sb, Sc and
S6, two from ‘EB’, S0 and S4, (Y arnane et a1. 2001) and the sweet cherry S4-
and S6-RNases .......................................................................... 57

Figure 3.2 Genomic blot analysis of ‘RS’, ‘EB’ and eleven progeny ....................... 59

Figure 3.3 Genetic map for the sour cherry linkage group 6 obtained with the ‘RS’ x
‘EB’ mapping population showing the location of the Sb-RNases (Sb) ........ 62

Figure B. 1: Graphic explanation of the expected S-genotypes and SI or SC phenotypes of
progeny in the Rheinische Schattenmorelle (RS) x Erdi B6terrn6 (EB)

population shown in Table A3 ..................................................... 144
Figure B.2: Schematic representation of the reciprocal inter-specific crosses between

sweet and sour cherry analyzed in Chapter 4 ..................................... 145
Figure B.3: Summary of the S-haplotypes in Rheinische Schattenmorelle ................ 149
Figure B.4: Summary of the S-haplotypes in Erdi Béterrnéi ................................. 150
Figure B.5: Summary of the S—haplotypes in Cigany 59 ..................................... 151
Figure B.6: Summary of the S-haplotypes in Surefire ........................................ 152
Figure B.7: Summary of the S—haplotypes in Montmorency ................................. 153
Figure B.8: Summary of the S—haplotypes in Ujfehértoi Fi'utds ............................. 154

CHAPTER 1

LITERATURE REVIEW

Introduction

Gametophytic self-incompatibility (SI) is a genetic mechanism that promotes out-
crossing in many flowering plants (de Nettancourt 1977). SI is controlled by a single
multi-allelic locus, called the S-locus, which is hypothesized to contain at least two genes
involved in determining the specificity of the SI interaction between the pollen tubes and
styles. The grth of pollen tubes is arrested within the style if the pollen and style
contain the same specificity alleles.

Sour cherry (Prunus cerasus L.) is an allotetraploid species produced by the
hybridization of the diploid sweet cherry (P. avium L.) and the tetraploid ground cherry
(P. fiuticosa Pall.) (Olden and Nybom, 1968). Whereas sweet cherry has a classic
gametophytic SI system, sour cherry segregates for SI and self-compatibility (SC) (Lech
and Tylus, 1983; Redalen 1984). The presence of SI sour cherry selections is unusual,
since SI typically breaks down as a result of polyploidy (Livermore and Johnstone 1940;
Crane and Lewis 1942; Stout and Chandler 1942; Brewbaker 1954; Pandey 1968). The
reason for the partial breakdown of SI in sour cherry is unknown. In the Solanaceae,
which has a similar gametophytic SI system, the breakdown of SI is apparently caused by
the competition of pollen-S products in heteroallelic pollen, i.e. pollen containing two
different pollen-S products.

For growers, SI sour cherry trees require mixed plantings with pollinator cultivars
in their orchard and the use of bees to ensure proper cross pollination and fruit set. The
need to use valuable orchard space for pollinators and the reliance on bees for adequate
fruit set compel growers to desire SC cultivars rather than SI ones. Thus, sour cherry

breeders are interested in breeding SC trees. Without markers for SC, the breeder may

have to wait three to five years after making a cross to determine if the tree is 81 or SC.
Therefore, the development of molecular markers for SC would facilitate sour cherry
breeding and reduce the number of seedlings that a breeder would need to keep.
However, without knowledge of the genetic control of SI and SC in sour cherry, it is
impossible to develop molecular markers that could be used to screen for SC seedlings.
The ultimate goal of this research was to determine the genetic control of SI and
SC in sour cherry to facilitate marker development. To reach this goal, it was first
necessary to test the hypothesis that gametophytic S1 in sour cherry involves the stylar S-
RNase as the style specificity component. This involved the determination of the S-
haplotypes in sour cherry and the relationship of these S—haplotypes to those in sweet
cherry. With these S-haplotypes defined, it was then possible to systematically conduct
genetic and genomic investigations of these S-haplotypes by themselves or in
combination. Finally, a hypothesis for the genetic control of SI and SC was developed

and verified in self-pollination and crossing experiments.

Literature Review

This literature review will focus on the genetic structure of sour cherry, the mechanism of

gametophytic self-incompatibility (SI), and the proposed mechanisms for the breakdown

of gametophytic SI.

The Origin of Sour Cherry

Sour cherry (Prunus cerasus L.) is an allotetraploid that was produced by the
hybridization of the diploid sweet cherry (P. avium) and the tetraploid ground cherry (P.
fiuticosa) (Olden and Nybom, 1968). This hybridization likely occurred multiple times
and in both directions, i.e. sweet cherry x ground cherry and ground cherry x sweet
cherry, as evidenced by the presence of both avium-type and fruticosa-type cytoplasm in
sour cherry (Brettin et al., 2000). The fi'uticosa-type cytoplasm is most prevalent but the
avium-type cytoplasm has been detected in some selections, such as Cigany 59. The
recurrent formation was aided by the overlapping distribution of sweet cherry and ground

cherry in Eastern Europe.

The Genomic Structure of Cherry

The genome size of sweet cherry is small (2C = 0.7pg; 338 Mb), approximately
double the size of the Arabidopsis thaliana genome (Arumuganathan and Earle, 1991).
The relatively small amount of repetitive DNA sequences in the Prunus genome made it
relatively easy for Prunus geneticists to identify a putative pollen-S gene (Lai et al., 2002;
Entani et al., 2003; Ushijima et al., 2003). Researchers working with more traditional
model systems such as tomato, petunia and tobacco were overwhelmed by the highly
repetitive nature of the Solanaceous S-locus (Coleman and Kao, 1992). However, the
inability to transform Prunus species makes it impossible to conduct the necessary gain-
of-function and loss-of-firnction transformation experiments to prove that a putative gene
is the true pollen-S gene. The first genetic map of sour cherry, consisting of RFLP

markers, was published in 1998 (Wang et al., 1998).

Although sour cherry is an allotetraploid that predominately exhibits disomic
inheritance, it also exhibits a low frequency of tetrasomic inheritance (~5%) (Beaver et

al. 1993) and quadrivalent pairing characteristic of an autotetraploid (Wang et al., 1998).

Prevalence of Self-Incompatibility

Self-incompatibility is one of the most prevalent mechanisms that prevents
inbreeding and promotes out crossing. Although many forms of SI exist, the S-RNase-
based gametophytic SI found in Prunus and the Solanaceae is one of the most widespread
and economically important.

Phylogenetic analysis of the S-RNase gene suggests that the S-RNase—based
gametophytic SI systems found in the Solanaceae, Scrophulariaceae and Rosaceae most
likely share a common origin (Igic and Kohn, 2001). The most recent common ancestor
of these three distantly related plant families is the ancestor to approximately 75 percent
of all dicot families.

Gametophytic SI exists in many Prunus species, including several diploid species
such as sweet cherry (Crane and Lawrence, 1929; Crane and Brown, 193 7; Way, 1967),
almond (P. dulcis: Socias i Company et al., 1976), Japanese apricot (P. mume) and plum
(P. domestica: Crane and Lawrence, 1929). A majority of the tetraploid sour cherry
individuals are SC; however, SI types can be found in the center of diversity (Lech and
Tylus, 1983; Redalen 1984). In addition, SI progeny can arise from crossing involving

two SC sour cherry selections (Lansari and Iezzoni, 1990).

Gametophytic Self-Incompatibility in Sweet Cherry

Early classification of S—haplotypes and incompatibility groups was done solely
via crossing experiments, which led to the identification of six S-haplotypes and 13
incompatibility groups (Mathews and Dow, 1969). Since the initial discovery that the
stylar product of the S-locus in sweet cherry is an S-RNase (Boskovic' and Tobutt, 1996)
the pace of S-haplotype classification and discovery has accelerated. Isoenzyme gels
(Boskovié and Tobutt, 2001), PCR profiles (Sonneveld et al., 2001; Wiersma et al., 2001;
Choi et al., 2002; Sonneveld et al., 2003), RFLP analyses (Tao et al., 1999) and cDNA
sequences (Tao et al., 1999; Wiersma et al., 2001) have been used to describe a total of

17 S-haplotypes.

The Mechanism of the RNase-Based Gametophytic Self-Incompatibility System

Gametophytic SI is controlled by a single multi-allelic locus, called the S-locus,
which is hypothesized to contain at least two genes involved in determining the
specificity of the SI interaction between pollen tubes and styles (de Nettancourt, 1977).
Because of the presence of multiple genes within the S—locus, the term “haplotype” has
been used to describe variants of the S-locus whereas the term “allele” describes variant
of a given polymorphic gene at the S-locus (McCubbin and Kao, 2000). The growth of
pollen tubes is arrested within the style if the pollen tube has a pollen-S product in
common with either of the two S-RNases in the style.

The stylar component of gametophytic SI is a ribonuclease, called S-RNase,
which was first discovered in the Solanaceae (Anderson, 1986; McClure et al., 1989),

followed by the Rosaceae (Sassa et al., 1993) and Scrophulaliaceae (Xue et al., 1996).

These S-RNases are expressed in the transmitting tract of styles and cause the
degradation of incompatible pollen tubes. S-RNases enter the pollen tubes of both
compatible and incompatible pollen (Luu et al., 2000) but only degrade rRNA of
incompatible pollen (McClure et al., 1990). Whether the S-RNase is activated in
incompatible pollen tubes or inactivated in compatible pollen tubes is unknown, although
the recent discovery that the pollen-S gene is an F-box gene (see below) suggests that the
S-RNase in compatible pollen tubes is quickly degraded by the ubiquitin/proteasome
proteolytic pathway (U shijima et al., 2003). Gain-of-firnction and loss-of-function
experiments have shown that S-RNase is necessary and sufficient for a style’s ability to
reject pollen (Lee et al., 1994; Murfett et al., 1994). In addition, mutation analyses have
shown that RNase firnction is necessary for the rejection of self-pollen (Huang et al.,
1994)

The S-RNase gene is composed of five conserved regions and two or one
hypervariable regions in the Solanaceae (Ioerger et al., 1991) and Rosaceae (U shijima et
al., 1998), respectively. HVa and HVb, the two hypervariable regions in Solanaceous S-
RNases, are exposed on the surface of the S-RNase protein (Ida et al., 2001) and under
positive selection (Ishimizu et al., 1998), suggesting that they are likely the determinants
of S-RNase specificity and may interact directly with the pollen-S product. Matton et al.
(1997) provided functional evidence by changing the specificity of an S-RNase simply by
swapping the HVa and HVb domains with those from a different S-RNase allele.
Rosaceous S-RNases only have a single hypervariable region (RHV), which corresponds

to the HVa from the Solanaceous S-RNases (U shijima et al., 1998).

The S-RNase gene in all Solanaceous and Rosaceous species, including Malus
species and Pyrus species but not Prunus species, contains a single intron (Igic and Kohn,
2001). The S-RNase gene from Prunus species contains a second intron near the 5' end
of the gene. The lengths of these introns vary greatly between S-haplotypes, making
them very useful for S-RNase genotyping (Tao et al., 1999; Wiersma et al., 2001).

The pollen component of gametophytic SI has, until recently, remained elusive.
Recently, several candidate genes have been hypothesized to be the pollen-S gene. In
each case, the candidate gene is a pollen-specific F-box gene that is located in the S-
locus. The first report of an F-box gene, named S-locus F-box gene (SLF), was from
Antirrhinum (Lai et al., 2002), which was later demonstrated to interact with the S—RNase
in a haplotype-specific manner (Qiao et al., 2004). Similar SLF genes were isolated from
Prunus mume (Entani et al., 2003). A different F -box gene, named S-haplotype-specific
F-box gene (SFB), was isolated from P. dulcis (U shijima et al., 2003) and showed higher
levels of polymorphism. Transformation is not possible in these species, making it
impossible to conduct the necessary gain-of-function and loss-of-function transformation
experiments to prove that one of these F-box genes is the true pollen-S gene. However,
correlation of mutations in the SFB with a loss-of-function of the pollen-S product has
provided strong evidence that SFB is the pollen-S gene (see “Proposed Mechanisms for
the Breakdown of gametophytic SI ”; Ushijima et al., 2004). More recently, an F-box
gene from Petunia (PiSLF) was shown via transformation to be sufficient for controlling

the S-haplotype specific interaction with the style indicating that it is the pollen-S gene

(Sijacic et al., 2004).

The finding that the pollen-determinant is an F-box protein suggests a possible
mechanism for the interaction between the S-RNase and the pollen-S product. F-box
proteins are components of SCF complexes, which regulate protein degradation in the
ubiquitin / proteasome proteolytic pathway (Deshaies, 1999). The F-box protein acts as a
protein receptor for the SCF, allowing the polyubiquitination and eventual degradation of
the protein by the 26S proteasome. Thus, SFB might form a complex with SCF that
polyubiquitinates all non-self S-RNases, thus causing their destruction. SFB might
interact with its cognate S—RNase in a different manner to prevent polyubiquitination and
allow it to remain active (Ushijima et al., 2003).

SFB is composed of many conserved residues, including a conserved region in the
N-terminus that makes up the F-box motif, two variable regions, and two hypervariable
regions, HVa and HVb (Ikeda et al., 2004). It is likely that the two hypervariable regions

play a role in the S-haplotype-specific interaction with cognate S-RNases.

Proposed Mechanisms for the Breakdown of Gametophytic SI

SI is believed to be the ancestral state in as many as 75 percent of dicot families
(Igic and Kohn, 2001). However, SI is not observed in all of these species, suggesting
the repeated breakdown of SI to create SC individuals, populations or species. There are
four main ways in which 81 can breakdown. These four mechanisms are described
below.

One way in which SC can arise in SI plants is through the mutation of the S-
RNase, the pistil determinant of the haplotype-specific rejection of pollen. These

mutations may either disrupt the fimction or the expression of the S-RNase in the styles.

Huang et al. (1994) were able to construct mutant S-RNase genes containing amino acid
substitutions at the RNase active sites. These mutations prevented function of the S-
RNase. Similar mutations in the wild would make the S-RNase non-functional, thus
resulting in the inability to reject pollen containing the cognate pollen-S product.

Yamane et al. (2003) reported the presence of a ~26OO bp insertion in the putative
promoter region of the sour cherry San-RNase. This insertion prevented expression of the
San-RNase in styles; however, it had no effect on the expression or fimction of the pollen-
S gene. Both of these types of mutations specifically disrupt the ability of a style to
recognize and degrade self-pollen. Pollen containing the S6m-haplotype could still be
rejected by styles containing a functional S6-RNase.

Alternately, mutations could occur in the pollen-S gene, thus making it incapable
of eliciting an SI reaction. Ushijima et al. (2004) reported structural mutations in SFB,
the putative pollen-S gene, of two pollen-part mutants (PPMs) from Prunus. A four base
pair deletion upstream from HVa and HVb in the S4v pollen-part mutant in sweet cherry
caused a frame-shift that results in defective SFB transcripts that lack the HVa and HVb.
A 6.8 kb insertion upstream from the HVa and HVb of the SfSFB in P. mume caused a
truncated SFB transcript which lacks the HVa and HVb regions. These mutations result
in a loss of pollen-S gene function; however, the S-RNase remains functional and capable
of rejecting pollen containing a corresponding functional pollen-S gene. Interestingly,
despite the identification of several PPMs through mutant screens, no PPMs caused by
either the loss of expression of the pollen-S gene or the expression of a mutant pollen-S
gene have been observed in the Solanaceae (Pandey 1967; van Gastel and de Nettancourt,

1975; Golz et al., 1999; Golz et al., 2000). Instead, all identified PPMs were caused by

10

the duplication of the pollen-S gene (see “fourth class of mutations” for more details).
This suggests that the loss of a functional pollen-S product does not cause a breakdown in
SI in the Solanaceae.

The previous two mechanisms involve mutations of the S-locus linked specificity
components of the SI reaction. However, mutations may occur in genes located outside
of the S-locus and result in SC. McClure et a1. (1999) detected a small asparagine—rich
protein, named HT, which is critical for the SI reaction, although it is not involved in the
initial interaction between the S-RNase and the pollen-S product. They report that
diminished expression of the HT gene is correlated with the breakdown of SI. Mutations
in “modifier genes”, such as HT, can cause the disruption of the interaction between the
S-RNase and pollen-S product of all S-haplotypes rather than just the loss-of-function of a
single S—haplotype.

A fourth class of mutations that can cause the breakdown of SI is the duplication
of the pollen-S gene. Two common mechanisms for the duplication of the pollen-S gene
are the creation of centric fragments containing the S-locus (Brewbaker and Nataraj an,
1960) and the increase in ploidy level. Crane and Lawrence (1931) were the first to
associate an increase in ploidy level with a conversion from S1 to SC while working with
sweet cherry. Later, similar observations were made in Pyrus communis (Crane and
Thomas, 1939), Solanum (Livermore and Johnstone, 1940), Petunia (Stout and Chandler,
1941), Nicotiana alata (Pandey 1968), and Lycopersicon peruvianum (de Nettancourt et
al., 1974). It was also observed that the breakdown was unilateral in the Solanaceous
plants, meaning that tetraploid styles maintained their ability to reject pollen from diploid

plants but pollen from tetraploid plants could not be rejected by diploid or tetraploid

11

styles (Livermore and Johnstone, 1940; Stout and Chandler, 1941; Crane and Lewis,
1942; Pandey, 1968; de Nettancourt et al., 1974; Chawla et al., 1997). Stout and
Chandler (1942) observed that this breakdown in polyploids occurred in heterozygous
plants but not homozygous ones, suggesting that the breakdown only occurs in
heteroallelic pollen. Lewis (1943) obtained similar results with Oenothera tetraploids and
suggested that a competition between different pollen-S products within a single pollen
tube causes the breakdown of SI in heteroallelic pollen but there is no competition
between S-RNases in styles. More recent experiments involving Petunia hybrida support
the hypothesis that heteroallelic pollen, but not pollen containing multiple copies of the
same pollen-S gene, loses its SI phenotype (Entani et al., 1999). The breakdown of SI in
heteroallelic pollen in the Solanaceae is so well accepted that transformation experiments
were recently designed to take advantage of this phenomenon to confirm that PiSLF is

the actual pollen-S gene (Sijacic et al., 2004).

12

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17

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18

CHAPTER 2

REVISITING THE S-ALLELE NOMENCLATURE IN
SWEET CHERRY (PRUNUS A VIUM) USING RFLP PROFILES

I9

Abstract

Correct assignment of self-incompatibility alleles (S-alleles) in sweet cherry (Prunus
avium L.) is important to assure fruit set in field plantings and breeding crosses. Until
recently, only six S-alleles had been assigned. With the determination that the stylar
product of the S-locus is a ribonuclease (RN ase) and subsequent cloning of the S-RNases,
it has been possible to use isoenzyme and DNA analysis to genotype S-alleles. As a
result, numerous additional S-alleles have been identified; however, since different
groups used different strategies for genotype analysis and different cultivars, the
nomenclature contained inconsistencies and redundancies. In this study restriction
fragment-length polymorphism (RF LP) profiles are presented using HindIII, EcoRI,
DraI, or XbaI restriction digests of the S-alleles present in 22 sweet cherry cultivars
which were chosen based upon their unique S-allele designations and/or their importance
to the United States sweet cherry breeding community. Twelve previously published
alleles (S 1, S 2, S3, S4, S5, S6, S7, S9, S10, S11, 512, and 513) could be differentiated by their
RFLP profiles for each of the four restriction enzymes. Two new putative S-alleles, both
found in ‘NY1625’, are reported, bringing the total to 14 differentiable alleles. We
propose the adoption of a standard nomenclature in which the sweet cherry cultivars
‘Hedelfingen’ and ‘Burlat’ are S 3S5 and S 3S9, respectively. Fragment sizes for each S-

allele/restriction enzyme combination are presented for reference in firture S-allele

discovery projects.

20

Introduction

Self-incompatibility (SI) is a common mechanism in flowering plants that
prevents self-fertilization and promotes out-crossing (de Nettancourt, 1977). In
gametophytic self-incompatibility (GSI), SI is determined by a single, multi-allelic locus,
called the S-locus in which the compatibility of a cross is determined by the haploid
genome of the pollen and the diploid genome of the pistil. In GSI, pollen tube growth is
arrested if the pollen tube has a S-allele in common with one of the two S-alleles in the
style. The S-locus is composed of multiple genes, one of which is an RNase (S-RNase)
that is expressed only in the pistil. A second gene that is hypothesized to be expressed
specifically in the pollen has yet to be determined from any GSI species.

Sweet cherry (Prunus avium) fertilization is controlled by a GSI system and
therefore, knowledge of the S-allele composition of a tree is crucial for compatible
pollination and fruit set. Knight (1969) named six S-alleles (S 1, S 2, S 3, S4, S 5, and S6) and
categorized the cultivars into 13 compatibility groups and a Group 0, which included
cultivars that were SI but able to pollinate cultivars in all the other groups. As is the case
with other reported GSI systems, the stylar S-allele component in 81 members of the
Rosaceae family is an S-RNase (Boskovic and Tobutt, 1996; Broothaerts et al., 1995;
Burgos et al., 1998; Ishimizu et al., 1996; Sassa et al., 1992; Sassa et al., 1996; Tao et al.,
1997; Tao et al., 1999; Tomimoto et al., 1996; Ushijima et al., 1998; Yamane et al.,
1999). In sweet cherry, RNase isoenzymes (Boskovic et al., 1997) and cDNA sequences

(Tao et al., 1999) have been associated with the stylar S-allele RNases.

21

Sour cherry (P. cerasus L.), which is a hybrid tetraploid species between sweet
cherry and ground cherry (P. fiuticosa Pall), consists of self-compatible and self-
incompatible individuals; however, unlike sweet cherry, control of SI in sour cherry is
unknown. Our long term goal is to determine the genetic control of SI in sour cherry
(Yamane et al., 2001). We hypothesize that a similar RNase stylar component is present
in sour cherry and that sweet and sour cherry may share common S-alleles. However,
before embarking upon S-allele discovery in sour cherry, we needed to have a clear
definition of the S-alleles that had been identified in sweet cherry.

A review of the sweet cherry S-allele literature revealed that potentially similar
sweet cherry S-alleles had been assigned differing nomenclature (Boskovic and Tobutt,
1996; Boskovic et al., 1997; Choi et al., 2000; Knight, 1969; Schmidt and Timmann,
1997; Schmidt et al., 1999; Tao et al., 1999; Tehrani and Lay, 1991; Wiersma et al.,
2001; Yamane et al., 2000). The confirsion seems to originate from the initial incorrect
classification of ‘Hedelfingen’ and ‘Burlat’ into Group VII with the assigned S-alleles,
S4S5 (Knight, 1969). Since ‘Hedelfingen’ and ‘Burlat’ are cross compatible, they should
have not been assigned to the same group. Tehrani and Lay (1991) recognized the S-
allele misclassification of ‘Hedelfingen’ and assigned it to Group 0. Boskovic et al.
(1997) proposed that ‘Hedelfingen’ contains the S 3- and S 5-alleles. Crosses done in
Germany confirmed that ‘Hedelfinger’ (‘Hedelfingen’) was S 3S 5 while ‘Burlat’ was
determined to contain neither S4 nor S 5 (Schmidt and Timmann, 1997). ‘Burlat’ was
assigned the S-alleles (S 3Sx) where S. represented a novel S-allele (Schmidt et al., 1999).
In this manuscript we propose the adoption of the S 3S 5 nomenclature for ‘Hedelfingen’ as

proposed by Boskovic et al. (1997), Schmidt and Timmann (1997) and Schmidt et a1.

22

(1999). Therefore the objective of this research was to use restriction fragment-length
polymorphisms (RF LPs) to characterize 12 sweet cherry S-alleles that had been published
previously, as well as to characterize two unique S-alleles, and to propose the adoption of
a standard nomenclature for these S-alleles. In addition, the S-genotypes of five new
cultivars important to the United States sweet cherry breeding community are reported.
Fragment sizes for each S-allele/restriction enzyme combination are also presented so this

information can be used as a reference in future S-allele discovery projects.

Materials and Methods

Plant material

Young leaf tissue was collected from 22 sweet cherry cultivars in the spring.
Leaves of ‘Napoleon’, ‘Cavalier’ and ‘Gold’ were collected at the Michigan State
University Northwest Horticultural Research Station, Traverse City, Mich. Leaves of
‘Charger’, ‘Gaucher’, ‘Inge’, and ‘Orleans 171’ were kindly provided by K. Tobutt (East
Malling, United Kingdom). Leaves of ‘Early Rivers’, ‘Burlat’, ‘Schneiders’, ‘Seneca’,
‘Valera’, ‘Hedelfingen’, ‘Nadino’, ‘NY1625’, and ‘Guigne d'Annonay’ were kindly
provided by C. Choi and R. L Andersen (Geneva, NY). Leaves of ‘Noble’ were kindly
provided by B. Lay (Vineland, Ontario, Canada). Leaves of ‘Chelan’, ‘Tieton’, PMR-l,
and PC-8007-2 were kindly provided by G. Lang (Prosser, Wash). Leaves of ‘Mona’
(DPRU 2046) were obtained from the US. Department of Agriculture National Clonal

Repository, Davis, Calif. Where possible, the leaf material was placed immediately on

23

dry ice. All frozen and fresh leaf material was lyophilized and stored at —20 °C until

needed for DNA isolation.

DNA isolation
Total DNA was isolated from young leaves using the CTAB method described by

Stockinger et al. (1996).

Genomic DNA blot analysis

Six micrograms of DNA was digested with either HindIII, EcoRI, DraI, or Xbal
(Boehringer Mannheim Biochemicals, Indianapolis, Ind.), run on 0.9 % agarose gel for
36 h at 30 V, and transferred to a nylon membrane (Hybond-N+, Amersham, Piscataway,
NJ.) according to Wang et al. (1998). Polymerase chain reaction (PCR) amplified
fragments of the S6-RNase cDNA from sweet cherry (Tao et al., 1999) were used as the
probe. Probes were radiolabelled with 32P-dCTP (DuPont, Boston) using the random
primer hexamer-priming method described by Feinberg and Vogelstein (1983). After
hybridization at 60 °C for 16 h and high stringency washes (2 X 30 min with 2X SSC and
1% SDS followed by 2 X 30 min with 0.2X SSC and 0.5% SDS at 60 °C), bound

radioactivity resulting fi'om hybridizations was detected with X-ray film.

PCR amplification of S-alleles

PCR was performed on the sweet cherry cultivars using two primer pairs: 81-19
(5' CCA CCG ACC AAC TGC AGA GT 3') / SI-20 (5' TGG TAC GAT TGA AGC GT

3'), and 81-31 (5' STT STT GST TTT GCT TTC TTC 3') / SI-32 (5' CAT AGG CCA

24

TGR ATG GTG 3') which were designed by Wiersma et al. (2001). The PCR conditions
were identical to those used by Wiersma et a]. (2001). PCR reactions were run in a DNA
Thermal Cycler 480 (Perkin Elmer, Norwalk, Conn), the resulting PCR mixtures were
run on 0.9% agarose gels, and the DNA bands were visualized by ethidium bromide

staining.

Results and Discussion

Twenty-two sweet cherry cultivars were analyzed by RFLP analyses using
HindIII, EcoRI, Dral, or XbaI restriction digestions (Figure 2.1). The four RFLP
analyses gave consistent results, and it was possible to distinguish 14 different putative S-
alleles with each of the four restriction enzymes (Table 2.1).

The S-genotypes of ‘Early Rivers’ (S 1S2), ‘Napoleon’ (S 3S4) and ‘Gold’ (S 3S6)
have not been questioned in the literature since first published (Knight, 1969) (Table 2.2).
The RFLP fragment sizes for the S1, S2, S 3, S4, and S6 alleles following HindIII and EcoRI
digests agree with those of Tao et al. (1999) with only slight variations due to enhanced
resolution since in the present study the fragments were separated for a longer period of
time on the agarose gel. In the HindIII digest, this enabled S1 to be distinguished from S 3
while S 2, S4, and S6 were distinguished from one another, and in the EcoRI digest, this
enabled S1 to be distinguished from S4. Each of these five S-alleles exhibits just one
fragment with the exception of the S4- and S 2- alleles, which exhibit two fragments

following HindIII and DraI digests, respectively (Table 2.1, Figure 2.1).

25

Figure 2.1. Genomic DNA blot analysis of 22 sweet cherry cultivars. Six micrograms of
Genomic DNA was digested by (A) HindIII, (B) DraI, (C) EcoRI, or (D) XbaI blotted to
membrane and hybridized to the cDNA encoding S6-RNase. Lambda/HindIII marker was
used for size determination. (a) 'Early Rivers' (S 1S2), (b) ‘Napoleon’ (S 3S4), (c) ‘Burlat’
(S 359), (d) ‘Gold’ (S 3S6), (e) ‘Charger’ (S 1S7) (f) ‘Gaucher’ (S 355), (g) ‘Inge’ (S459), (h)
‘Orleans 171’ (S109, 1), (i) ‘Schneider’ (S3512), (j) ‘Mona’ (S 3S9), (k) ‘Seneca’ (S1S5), (I)
‘Valera’ (S 155), (m) ‘Hedelfingen’ (S 355), (n) ‘Nadino (S 3S 5) (o) ‘NY1625’ (SuSv), (p)
‘Guigne d'Annonay’ (S257), (q) ‘Chelan’ (S 3S9), (r) ‘Tieton’ (S 3S9) (s) ‘PMR-l’ (S4S9), (t)

‘8007-2’ (S4S9), (u) ‘Cavalier’ (S2S3), and (v) ‘Noble’ (S6813).

26

A HindIII
k l m no

    

abcdefghij

   

~23
. —2.0

 

B DraI
abcdefghij klmnopqrstuv

 

27

C EcoRI
abcdefghjklmnopqrstu

\ .

    

 

 

 

28

Table 2.1 Sizes of DNA restriction fragments for sweet cherry S—alleles used in this study

 

 

 

 

Size (kb)
S-allele HindIII EcoRl DraI XbaI
1 8.7 1.5 2.5 13.0
2 5.6 4.4 0.8, 1.0 2.6
3 8.8 13.1 2.3 20.0
3 ‘Gaucher’ z 8.8 13.1 2.3 20.0
4 5.6, 6.1 1.8 1.8 8.8
5 9.4 3.5 5.3 6.8
6 5.8 11.0 3.5 5.5
7 3.5, 5.8, 8.7 3.3, 6.0 0.8, 3.45 21.0
9 3.1 7.9 0.9 0.0, 16.0, 18.0
9 ‘Burlat’ y 3.1, 4.0 7.9 06", 0.9, 1.2 15.0
10 or 11x 3.5, 5.8, 6.4, 6.6, 12.1 3.3, 5.0, 5.5 0.8, 1.6, 3.5 9.4, 13.0, 21.0
12 12.1 - 1.5, 1.9 16.0
13 4.6, 6.5 - 4,4 -
u or vw 2.5, 6.4 4.8 1.6, 2.7 -

 

‘ This S-allele in ‘Gaucher’ was originally thought to be a unique S-allele (S3) (Boskovic
et al. 1997).

y This S-allele in ‘Burlat’ was originally thought to be a unique S-allele (Sx) (Schmidt et
al,1999)

" These are the two S-alleles in ‘Orleans 171’. Restriction fragments for S10 and S 1 1 were
grouped together because it could not be determined which fragments
corresponded to each S-allele.

w These are the two putative unique S-alleles in ‘NY1625’. Restriction fragments for S“

and S. were grouped together because it could not be determined which fragments
corresponded to each S-allele.

29

Table 2.2 S-allele genotypes of 17 sweet cherry cultivars used in this study.

 

 

 

Cultivar S-allele genotype Other published nomenclature
‘Early Rivers’ 1,2 2

‘Napoleon’ 3,4 2

‘Hedelfingen’ 3,5 3”“ 4,5 2 ; 3,x w ; 3,15 V
‘Nadino’ 3,5 " 3,x “’
‘Seneca’ 1,5 1,x w

‘Valera’ 1,5 "“ 1,x w; 1,15 V
‘Gold’ 3,6 ‘

‘Charger’ 1,7 ‘

‘Guigne d’Annonay’ 2,7 s 2,2 w
‘Gaucher’ 3,5 3 5,8 ‘

‘Inge’ 4,9 ‘

‘Orleans 171’ 10,11 ‘

‘Schneiders’ 3,12 5 3,13 V ; 3,y w
‘Burlat’ 3,9 ’ 3,x " ; 4,5 2 ; 3,5 w’”
‘Mona’ 3,9 2,14 "
‘Noble’ 6,13 s 6,? v , 1,6 w .
‘NY1625’ u,v 4,x w

7‘ Knight (1969).

y Boskovic and Tobutt (1996).

" Schmidt et al. (1999).
“' Choi et al. (2000).

" Wiersma et al. (2001).
“ Way (1968)
‘Boskovié et al. (1997).

’ Boskovic and Tobutt (2001).

' Tao et al. (1999).

30

As reported by Boskovic et al. (1997), ‘Charger’ (S 1S 7) and ‘Inge’ (S459) each
exhibit one new S-allele. These alleles, called S 7 and S9, displayed from one to three
unique fragments per allele following Southern hybridization (Table 2.1, Figure 2.1).
PCR of the S7 allele using the primer pair SIl9/S120 did not amplify any fragment;
whereas, a fragment of 425 base pairs (bp) (similar to the S 2, S9, and $12 alleles) was
amplified when using the primers SI31/SI32 (Figure 2.2). S9 produced one 745 bp
amplification product and two fragments of 425 bp (similar to 52, S7, and S12 alleles) and
615 bp, respectively, when amplified with SIl9/S120 and 8131/8132.

The presence of two unique S-alleles in ‘Orleans 171’ (S MS 1 1) also agrees with
that of Boskovic et al. (1997). RFLP analysis of ‘Orleans 171’ produced either three or
five fragments, depending on what restriction enzyme was used (Table 2.1, Figure 2.1)
and the fragment patterns did not match that of any known S—alleles. It could not be
determined which RF LP fragments represent the S10 versus the S11 alleles since
differential cultivars, such as S3510 and S 3S 1 1 are not available. Neither S10 nor S11 could
be amplified using either of the PCR primer pairs, SIl9/S120 or S13 1/SI32 (Figure 2.2).
These results support the conclusion that ‘Orleans 171’ contains two unique S-alleles.

‘Noble’ was initially assigned the S-alleles S 1 and S6 (Choi et al., 2000), but was
later found to contain 56 and a unique S-allele, which was temporarily named S 2
(W iersma et al., 2001). Restriction digestion of ‘Noble’ with either HindHI or DraI
produced fi'agments that did not match any found in other cultivars (Table 2.1, Figure
2.1). Boskovic and Tobutt (2001) named this allele S13. In order to remain consistent

with the European nomenclature, we suggest retaining the genotype of $6513 for ‘Noble’.

31

A 3119—3120
abcdefghijk

 

B SI31—SI32
abcdefg hijk

 

Figure 2.2. PCR analysis of S—alleles from sweet cherry. Genomic DNA was PCR
amplified using two primer sets: (A) SIl9/20 and (B) SI31/32. A 123 bp DNA ladder
was used for size determination. (a) ‘Early Rivers’ (S 1S2), (b) ‘Napoleon’ (S 3S4), (c)
‘Burlat’ (S 3S9), ((1) ‘Gold’ (S 3S6), (e) ‘Charger’ (S 1S7) (f) ‘Gaucher’ (S 3S 5), (g) ‘Inge’
(S4Sg), (h) ‘Orleans 171’ ($103“), (i) ‘Schneider’ (S3512), (j) ‘Mona’ (S 3S9), and (k)
‘Hedelfingen’ (5355).

32

The S-genotypes of the other cultivars in Table 2.2 have been more difficult to
determine and much of this difficulty can be traced to the initial misclassification of
‘Hedelfingen’ and ‘Burlat’ (Knight, 1969). All authors agree that ‘Hedelfingen’ and
‘Nadino’ have a S 3 allele and that ‘Seneca’ and ‘Valera’ have an S 1 allele. It is the
second common allele present in these four selections that has been assigned conflicting
nomenclature. Choi et al. (2000) called this allele 5... Wiersma et al. (2001) have named
this ‘Hedelfingen’ allele S15. Both groups of researchers called the ‘Burlat’ allele S 5.
However, both Boskovic et al. (1997) and Schmidt et al. (1999) called the allele in
‘Hedelfingen’ S5 prior to either of these publications. Therefore, we recommend that S 5
be adopted as the standard nomenclature for the allele present in ‘Hedelfingen’ and also
in ‘Nadino’, ‘Seneca’, ‘Valera’, and ‘Gaucher’ (Table 2.2). This S5 allele exhibited just
one fragment when digested with any of the four restriction enzymes (Table 2.1, Figure
2.1).

The unique S-allele present in ‘Burlat’ that was called S5 (Choi et al., 2000; Tao et
al., 1999; Wiersma et al., 2001) and S. (Schmidt et al., 1999) should be renamed.
Recently, this S-allele has been sequenced, and found to have an identical sequence to the
S9 allele found in ‘Inge’ (T. Sonneveld, personal communication). Therefore, we propose
the S-allele nomenclature for ‘Burlat’ be S 3S9. The RFLP profiles of the S9 allele in
‘Burlat’ are similar to the profiles of the S9 allele from ‘Inge’ following digestion with
HindIH, EcoRI, and DraI with the only differences being the presence of extra faint
bands in the ‘Burlat’ allele when digested with HindIII or DraI (Table 2.1, Figure 2.1).
This can be explained by differential length of exposure or differing amounts of DNA in

the digestion reaction. However, the profiles of the S9 alleles in ‘Burlat’ and ‘Inge’ are

33

significantly different after digestion with XbaI. This presents a shortcoming of RFLP
analysis for S-allele genotyping. If the restriction enzyme cut site(s) resulting in the
polymorphism(s) are not within the S-RNase, but instead are located in the regions
flanking the S-RNase, it is possible that identical S-alleles could exhibit different RFLP
fragments.

‘Mona’ used in this research has the same genotype as ‘Burlat’ (S3S9) (Figures 2.1
and 2.2). This contradicts the finding by Wiersma et a1. (2001) that the S-genotype of
‘Mona’ is S2S14. The probable explanation is that the ‘Mona’ trees from which the leaves
were collected for each study (the USDA Clonal Repository, Davis, Calif. and Vineland,
Ontario, Canada, respectively) were not the same cultivar. These conflicting results
reinforce the importance of not only knowing the purported cultivar, but also the source
in case identities are mistaken across locations.

Using PCR data, Choi et al. (2000) determined that ‘Guigne d’Annonay’
contained an allele that differed from any previously reported S-allele and thus named it
S2. The HindIII, EcoRI, DraI, and XbaI RFLP analyses all suggest that the S, allele in
‘Guigne d'Annonay’ is the same as the S7 allele in ‘Charger’ (compare lanes e and p on
Figure 2.1). Therefore, we propose that the actual S-genotype of ‘Guigne d'Annonay’
should be S 2S 7 (Table 2.2). Similarly, Yamane et al. (2000) used RFLP analyses and S-
RNase patterns on 2D-PAGE to discover a novel S-allele in the cultivar, Hinode (syn.
Early Purple). Based on their RFLP patterns after digestion with EcoRI or HindIII
restriction enzymes, this novel S-allele also appears to be S7.

‘Schneiders’ has been reported to contain an additional unique S-allele, named Sy

by Choi et a]. (2000), 513 by Wiersma et al. (2001), and S12 by Boskovic and Tobutt

34

(2001). In order to remain consistent with the European nomenclature, we suggest
retaining the genotype of S 3S 1 2 for ‘Schneiders’. S12 exhibited two fragments following
DraI digest and one fragment following HindIII and XbaI digests (Table 2.1).

Boskovic et al. (1997) presented the RNase isoenzyme patterns for five new S-
alleles: S7, Sg, Sg, S10, and S11. Whereas our RFLP and PCR analyses support the
conclusion that S7, S9, S10, and S11 are unique S-alleles, these analyses did not provide
evidence that S8, which is supposedly present in ‘Gaucher’, is truly a new S—allele. None
of the four restriction digests were able to detect a difference between S 3 and S8 (Table
2.1, Figure 2.1). PCR amplification using the primer pairs 8119/8120 and SI31/SI32 was
also unable to differentiate between S3 and S3 (Figure 2.2). Both the S 3 and S3 alleles
produced a fragment of 825 or 300 bp when amplified with SIl9/ S120 or S13 l/SI32,
respectively. Recent cloning and sequencing of the Sg allele has shown that the sequence
for the S 3 and S3 RNases are identical (Sonneveld et al., 2001). Therefore, the S-allele
designation for ‘Gaucher’ should be S 3S5 rather than S 5S8.

The RF LP patterns from the HindIII, EcoRI, and DraI RFLP analyses suggest that
the selection ‘NY1625’ contains two S-alleles represented by one fi'agment each that are
not found in any other cultivar (lane “0” in Figure 2.1A, B and C; Table 2.1). PCR
amplification of ‘NY1625’ using the primer pair SIl9/ S120 confirms the presence of at
least one unique S-allele in ‘NY1625’ as a single 670 bp fragment (data not presented)
was produced, which is different from all known S-alleles. However, using PCR, Choi
et al. (2000) reported that the S-genotype for this selection was 5er (S455, using the
proposed nomenclature), which would be expected given the parentage of ‘NY1625’

(‘Hedelfingen’ S 3S 5 x ‘Emperor Frances’ S 354) (Choi, 1999). It is possible that the DNA

35

sample contained some polysaccharide that altered the efficiency of the restriction digests
and PCR amplifications. However, it is also likely that the DNA used in these RFLP
analyses was mistakenly not collected from ‘NY1625’. We propose that these two S-
alleles be temporarily named Su and S. until crossing data confirms that they are indeed
unique S-alleles.

Table 2.3 shows the S—genotypes of four new selections from Washington State
University and a new cultivar from Michigan. Both ‘Chelan’ and ‘Tieton’ are S 359, while
PMR-l and PC-8007-2 are S4Sg. Choi et al. (2000) reported that the S-genotype of
‘Chelan’ is identical to that of ‘Burlat’, which agrees with the RFLP data. The other
three cultivars/selections have not been genotyped previously. PMR-l has been
confirmed to be SI (G. Lang, personal communication), thus, this breeding line must
contain S; as opposed to the fertile S-allele, S4'. The pedigree of PC-8007-2 suggests that
it contains the S4, allele; however, crossing data suggests that it is SI. These conflicting
results make it currently impossible to determine if PC-8007-2 contains S4 or S4,.
Currently, there are no molecular methods to differentiate between S4 and S41. Therefore,
more crossing data are needed to determine if PC-8007—2 is SI or SC. The genotype of
‘Cavalier’ is S 2S 3. The pedigrees for each of these new cultivars confirm the S-genotypes
determined by RFLP and listed in Table 2.3.

PCR has been used by several researchers to differentiate between S-alleles. Tao
et al. (1999) was able to differentiate between six S-alleles using two primer sets.
Likewise, Wiersma et a]. (2001) could distinguish nine S-alleles using two primer sets,

with the aid of restriction digestions of the PCR-amplified products. However, these

36

Table 2.3. S-genotypes for five Washington State University

and Michigan sweet cherry selections.

 

 

Selection S-allele genotype
‘Chelan’ 3,9
‘Tieton’ 3,9
PMR-I 4,9
PC-8007-2 4,9
‘Cavalier’ 2,3

 

37

studies did not examine all known S-alleles. In the current study, additional S—alleles
were examined. Some of the new S-alleles produced amplification fragments that were
the same size as those produced by previously studied S—alleles. For example, the

S13 1/SI32 primer pair could not distinguish between S2, S7, S9, and S 2 2 (Figure 2.2). In
addition, neither primer set could amplify S10 or S“. It is possible that restriction
digestion of the PCR products would allow for differentiation between S2, S 7, S9, and S12;
however, the digestions would be of no use to identify S10 and S 2 2, since they are both
null alleles. As more S-alleles are discovered, the number of null alleles and confounding
alleles is likely to increase. Nonetheless, PCR is still a useful tool for obtaining quick
confirmation of what S-alleles are in the progeny of a cross between known parents. In
addition, once new S-alleles are discovered and cloned, allele-specific primers could be
produced which would allow differentiation of all S-alleles by PCR for genotyping
projects. However, for S-allele discovery projects, a more powerful method for
differentiating between S-alleles is needed. The potential of RFLP for discovery and
identification of new S-alleles has been demonstrated by the fact that all S-alleles can be
distinguished based on their unique banding patterns after digestion with any of the four
restriction enzymes used in the present study. However, when interpreting RFLP data, it
must be taken into consideration that the S-allele probe is hybridizing to fragments that
include regions flanking the S-RNase. Ifthe flanking regions of two identical S-RNases
differ for their restriction enzyme cut sites, it is possible that different RFLP profiles may
be observed in the cultivars even though the S-alleles are not unique, leading to the

incorrect assumption that a new S-allele has been discovered.

38

Correct identification of S-allele genotype is critical for determining pollen
compatibilities for field plantings and breeding crosses. Less than 5 years ago, the
presence of only six S-alleles had been reported in sweet cherry (Boskovic and Tobutt,
1996; Schmidt and Timmann, 1997). Since the stylar component of the S-locus in sweet
cherry is believed to be an S-RNase, new methods are available to discover new S-alleles
and S-allele discovery has proceeded at a rapid pace. It would not be surprising if many
more unique S-alleles exist in natural populations of sweet cherry, since other plant
species having a GSI system have been reported to have a very large number of S-alleles.
For example, 37 and 39 different S-alleles were reported for evening primrose
(Oenothera organensis Munz) (Emerson, 193 9) and white clover (T rifolium repens L.)
(Atwood, 1944), respectively.

The first six S-alleles from sweet cherry were discovered using crossing data.
Since then, all of the subsequent S-alleles have been identified using molecular
techniques. However, proper verification that each S-allele is unique requires crossing
data. Unfortunately, as the number of unique S-alleles in sweet cherry increases, it
becomes more cumbersome to perform all of the necessary diallele crosses. As a result,
it is important to have available a molecular technique that can differentiate reliably
between all unique S-alleles. Perhaps the most accurate method to verify the uniqueness
of S-alleles, besides crossing, is to compare the amino acid sequences of each S-allele.
However, sequences have not been reported for all known S-RNases. The complete
amino acid sequences of S1 (AB028153), S2 (AJ298311), S3 (AB010306), S4
(AB028154), and S6 (AB010305) can be found on GenBank

(http://www.ncbi.nlm.nih.gov/). Until all amino acid sequences are reported, we propose

39

the use of RFLP analysis to investigate the uniqueness of an S-allele. For this to be
effective, it is necessary that the RFLP patterns for new S-alleles be published for
comparison. For example, Table 2.1 presents the fragment sizes for each of the S-alleles
as determined by RF LP analysis with HindIII, EcoRI, DraI, or XbaI restriction enzymes.
When a putative new S-allele is discovered, the researcher can compare its fragment sizes
with those presented in Table 2.1 to determine if the S-allele is likely a new allele, or if it
matches an already existing S-allele. The alternative to comparing data with that
presented in this table is to include all known S—alleles as controls, but as the number of
unique S-alleles increases, this will get more cumbersome. This strategy of comparing
RFLP fragments with fragments produced for known S-alleles in sweet cherry was used

to propose five new S-alleles in sour cherry (Yamane et al., 2001).

40

References

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Boskovic R, Tobutt KR (1996) Correlation of stylar ribonuclease zymograms with
incompatibility alleles in sweet cherry. Euphytica 90:245-250.

Boskovic' R, Tobutt KR (2001) Genotyping cherry cultivars assigned to incompatibility
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Broothaerts W, Janssens GA, Proost P, Broekaert WF (1995) cDNA cloning and

molecular analysis of two self-incompatibility alleles from apple. Plant Mol Biol
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Choi C (1999) On the genetics of self-incompatibility in sweet cherry: Identification of S-
genotypes; genetical and physiological effects on self-fertility and breakdown of
self-incompatibility. PhD Diss, Dept of Hort Sci, Cornell Univ, Ithaca, NY.

Choi C, Livermore K, Andersen RL (2000) Sweet cherry pollination: Recommendation
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de Nettancourt D (1977) Incompatibility in angiosperrns. Springer, New York.

Emerson S (193 9) A preliminary survey of the Oenothera organensis population.
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Identification and partial amino acid sequences of seven S-RNases associated with

self-incompatibility of Japanese pear, Pyrus pyrifolia Nakai. J Biochem 120:326-
334.

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Prunus. Eastern Press, London.

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Sassa H, Hirano H, Ikehashi H (1992) Self-incompatibility—related RNases in styles of
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Sassa H, Nishio T, Kowyama Y, Hirano H, Koba T, Ikehashi H (1996) Self-
incompatibility (S) alleles of the Rosaceae encode members of a distinct class of
the T2/S ribonuclease superfamily. Mol Gen Genet 250:547-557.

Schmidt H, Timmann EM (1997) On the genetics of incompatibility in sweet cherries 1.
Identification of S alleles in self-incompatible cultivars. Gartenbauwissenschaft
62:102-105.

Schmidt H, Wolfram B, Boskovic R (1999) Befruchtungsverhaltnisse bei Si’rB kirschen.
Erwerbsobstbau 41:42-45 (in German).

Sonneveld T, Robbins TP, Boskovic R, Tobutt KR (2001) Cloning of six cherry self-
incompatibility alleles and development of allele-specific PCR detection. Theor
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Identification of stylar RNases associated with gametophytic self-incompatibility in
almond (Prunus dulcis). Plant Cell Physiol 38:304-311.

Tao R Yamane H, Sugiura A, Murayama H, Sassa H, Mori H (1999) Molecular typing of
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using RFLP markers. Theor Appl Genet 97:1217—1224.

42

Way R (1968) Pollen incompatibility groups of sweet cherry clones. Proc Am Soc Hort
Sci 92:119-123.

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incompatibility groups by PCR and sequencing analysis. Theor Appl Genet
102:700-708.

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69:29-34.

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43

CHAPTER 3

SELF-COMPATIBILITY AND INCOMPATIBILITY
IN TETRAPLOID SOUR CHERRY (PRUNUS CERASUS L.)

44

Abstract

Gametophytic self-incompatibility (GSI) typically “breaks down” due to polyploidy in
many Solanaceous species, resulting in self-compatible (SC) tetraploid individuals.
However, sour cherry (Prunus cerasus L.), a tetraploid species resulting from
hybridization of the diploid sweet cherry (P. avium L.) and the tetraploid ground cherry
(P. fruticosa Pall.), is an exception, consisting of both self-incompatible (SI) and SC
individuals. Since sweet cherry exhibits GSI with 13 S-ribonucleases (S-RNases)
identified as the stylar S-locus product, the objectives were to compare sweet and sour
cherry S-allele firnction, S-RNase sequences and linkage map location as initial steps
towards understanding the genetic basis of SI and SC in sour cherry. S-RNases from two
sour cherry cultivars that were the parents of a linkage mapping population were cloned
and sequenced. The sequences of two S-RNases were identical to those of sweet cherry
S-RNases, whereas three other S-RNases had unique sequences. One of the S-RNases
mapped to the Prunus linkage group 6, similar to its location in sweet cherry and almond,
whereas two other S-RNases were linked to each other but were unlinked to any other
markers. Interspecific crosses between sweet and sour cherry demonstrated that
gametophytic SI exists in sour cherry and that the recognition of common S-alleles has
been maintained in spite of polyploidization. It is hypothesized that self-compatibility in
sour cherry is caused by the existence of non-functional S-RNases and pollen S-genes that

may have arisen from natural mutations.

45

Introduction

Self-incompatibility (SI) is a common evolutionary strategy used by flowering
plants to prevent self-fertilization and promote out-crossing (de Nettancourt 1977). In
gametophytic self-incompatibility (GSI), S1 is determined by a highly multi-allelic locus,
called the S-locus, in which the compatibility of a cross is determined by the haploid
genome of the pollen and the diploid genome of the pistil. In GSI, pollen tube growth is
arrested if the pollen tube has an S-allele in common with one of the two S-alleles in the
style. The S-locus has been classically described as a complex containing multi-allelic
genes expressed by the pollen and style and tight linkage between these components.
Because of the presence of at least two multi-allelic genes, the term “haplotype” has been
used to describe variants of the S-locus and the term “allele” to describe variants of a
given polymorphic gene at the S-locus (McCubbin and Kao 2000). In the Solanaceae and
the Rosaceae, the gene controlling the pistil’s self-incompatibility response is a
ribonuclease (S-RNase) which is expressed only in the pistil (McClure et al. 1989; Sassa
et al. 1992; Lee et al. 1994; Murfett et al. 1994; Broothaerts et al. 1995; Boskovic and
Tobutt 1996; Ishimizu et al. 1996; Sassa et al. 1996; Tomimoto et al. 1996; Tao et a1.
1997; Burgos et al. 1998; Ushijima et al. 1998; Tao et al. 1999; Yamane et al. 1999). A
second gene that is hypothesized to be expressed specifically in the pollen has yet to be
determined from any GSI species. Additional modifier genes have also been
demonstrated to be required for normal SI function (McClure 1999).

G81 present in diploid species has been observed to ‘break down’ due to

polyploidy with the tetraploid relatives frequently self-compatible (SC) (Livermore and

46

Johnstone 1940; Stout and Chandler 1942; Crane and Lewis 1942; Brewbaker 1954;
Pandy 1968; de Nettancourt et al. 1974; Ueda and Akimoto 2001). To explain this
phenomenon, Lewis (1947) proposed that pollen containing two different S-loci loses its
SI phenotype resulting in SC polyploid individuals. Evidence obtained from recent
research in Solanaceous species supports this theory (Chawla et al. 1997; Entani et al.
1999; Golz et al. 1999; Luu et al. 2001). In contrast, the GSI diploid sweet cherry
(Prunus avium L., 2n=2x=16) and the tetraploid sour cherry (P. cerasus L., 2n=4x=3 2)
represent a natural diploid — tetraploid series where the tetraploid individuals can be
either S1 or SC.

Sweet cherry and the tetraploid ground cherry (P. fruticosa Pall, 2n=4x=32) are
believed to be the parental species that gave rise to sour cheny multiple times via
unreduced gametes from sweet cherry (Olden and Nybom 1968; Iezzoni and Hancock
1984; Brettin et al. 2000). Although the vast majority of sour cherry cultivars are SC,
numerous SI cultivars exist in Eastern Europe, the center of diversity (Lech and Tylus
1983; Redalen 1984a, 1984b; Lansari and Iezzoni 1990; Iezzoni et al. 1990). However,
the SI phenotype is not limited to landrace cultivars as S1 sour cherry selections can result
from crosses between two SC sour cherry parents (Lansari and Iezzoni 1990). For
example, a sour cherry linkage mapping population generated by crossing two SC sour
cherry cultivars, ‘Rheinische Schattenmorelle’ (RS) x ‘Erdi B6term6’ (EB), segregates
for SI and SC (Wang et al. 1998). Since any successful new sour cherry cultivar would
have to be SC to avoid the production problems associated with providing pollinator
trees, our goal was to determine the genetic basis of SI and SC in sour cherry to increase

the likelihood of obtaining SC progeny in our sour cherry breeding program.

47

Sweet cherry exhibits classical gametophytic self-incompatibility with 13 S-
RNases identified and validated in crossing experiments (Matthews and Dow 1969;
Boskovic and Tobutt, 1996; Choi et al. 2000; Boskovic and Tobutt 2001; Hauck et al.,
2001; Wiersma et al. 2001). In contrast, there is only one study of SI in sour cherry that
takes advantage of the ability to determine putative S-RNase genotypes. Yamane et al.
(2001) recently cloned two S-RNases from ‘EB’; one matched the S4-RNase previously
cloned from sweet cherry and the second S-RNase was a novel S-RNase not previously
identified in sweet cherry. RFLP and PCR analysis of S-RNase alleles in a set of sour
cherry cultivars identified an additional four S-RNases that are presumably identical to
previously identified sweet cherry S-RNases and an additional three putative novel S-
RNases. The ‘RS’ and ‘EB’ mapping parents had the putative S-RNase designations,
SaSbSch and SaSrng, respectively. Yamane et al. (2001) further compared the S-RNase
allele composition of six SI with seven SC selections and found that all SI selections,
similar to the SC selections, contained three or four different putative S-RNase alleles.
This suggests that heteroallelic pollen alone may be insufficient to cause SC in tetraploid
sour cherry.

Due to the evolutionary relatedness of sweet and sour cherry, and the potentially
on—going gene flow between the two species, it is not surprising that S-RNases
presumably identical to those found in sweet cherry were identified in sour cherry
(Yamane et al. 2001). We further investigated 81 and SC in sour cherry by taking
advantage of the potential commonalities between these two species. The inheritance and
linkage map locations of the putative S-RNases fi'om ‘RS’ and ‘EB’ could also be

determined and compared with information from other Prunus species. In sweet cheny

48

and almond (Prunus dulcis), the S-locus has been mapped to the end of the Prunus
linkage Group 6 (Ballester et al. 1998; K. Tobutt, pers. comm.)

Our objectives were: (1) to determine if the S-alleles that appeared to be common
between sweet and sour cherry exhibited the expected recognition reactions in the styles
by making inter-specific crosses, (2) to determine the amino acid sequences of the S-
RNases in ‘EB’ and ‘RS’ and compare them with previously sequenced sweet cherry S-

RNases, and (3) to determine the sour cherry linkage map locations for the S-RNase loci.

Materials and Methods

Plant material

The two SI sour cherry cultivars ‘Crisana’ and ‘Tschemokorka’ were chosen for
pollination with sweet cherry cultivars based on previous examination of their S-RNase
profiles using PCR and RFLP analyses (Yamane et al. 2001). ‘Crisana’ contains three
different S-RNases, one of which is presumably present in double dose. Two of the S-
RNases produce RFLP and PCR fragment profiles identical to the S 2- and S4-alleles in
sweet cherry. The third S-RNase is not similar to any sweet cherry S-RNase, and is called
Sd. ‘Tschernokorka’ also contains three different S—RNases, only one of which is
identical to a sweet cherry S-RNase (S9). The other two S-RNases are named S0 and Sc.
The three sweet cherry cultivars used were ‘Schmidt’ (S2S4), ‘Rainier’ (S 1S4) and ‘Sato
Nishiki’ (S 3S6). ‘Crisana’, ‘Tschernokorka’, and ‘Schmidt’ are maintained at MSU’s

Clarksville Horticultural Experimental Station (CHES), Clarksville, Mich. ‘Rainier’ is

49

maintained at the North West Horticultural Research Station, Traverse City, Mich, while
‘Sato Nishiki’ pollen was collected from trees growing at the Experimental Farm of
Kyoto University, Kyoto, Japan, dried and frozen at -—20 C. All pollen samples were
tested to verify pollen viability prior to the crossing experiments as described by
Brewbaker and Kwack (1963).

A pseudo-testcross mapping population consisting of 85 progeny from the cross
between two sour cherry cultivars, ‘Rheinische Schattenmorelle’ (‘RS’) x ‘Erdi B6term6’

(‘EB’) (Wang et al. 1998) was maintained at CI-IES.

Analysis of pollen tube growth

Eight inter-specific crosses were performed and pollen tube growth was observed
to determine whether the crosses were compatible or incompatible. Styles from each of
the sour cherry cultivars (‘Crisana’ and ‘Tschernokorka’) were pollinated with pollen
from the sweet cherry cultivars, ‘Sato Nishiki’ or ‘Rainier’. Styles from the sweet cherry
cultivars (‘Rainier’ and ‘Schmidt’) were pollinated with ‘Crisana’ and ‘Tschernokorka’
pollen. Pollination tests were performed as described by Lansari and Iezzoni (1990) with
slight modifications. Pollen from newly opened flowers was collected from each of the
pollen parents. Pollen was dried for 24 h. For each of the stylar parents, a branch with
flowers at the balloon stage was brought into the laboratory and twenty flowers were
emasculated. All other flowers were removed. Ten emasculated flowers were hand
pollinated when receptive (24 h after emasculation) with each of the pollen sources. The
pollinated pistils were collected 72 h after pollination, placed in fixing solution [(1

chloroform : 3 (95%) ethanol : 1 glacial acetic acid) (v/v)] for 24 h, transferred into 100%

50

ethanol, and stored at 4 °C until used for observation. The pistils were washed
thoroughly under running tap water, incubated in 10 N NaOH for 5 to 6 h to soften the
tissues, and soaked in 0.1 % aniline blue solution with 33 mM K3PO4 for 1 h to
fluorescently stain the pollen tubes. Pollen tubes were observed by ultraviolet fluorescent
microscopy (BX60, Olympus, Tokyo, Japan).

To determine whether the 77 flowering progeny of the ‘RS’ x ‘EB’ mapping
population progeny were S1 or SC, in vitro pollination tests were performed as described
above. Styles from each of the progeny were pollinated with either self-pollen or with

pollen from a collection of several unrelated sour cherry cultivars (out-cross pollen).

cDNA Cloning of S-RNases

Total RNA was isolated from ‘RS’ styles as described by Tao et al. (1999). One
microgram of total RNA was used for first strand cDNA synthesis by SUPER SCRIPT 11
RT (Life Technologies, MD) with an Adp-dT primer (5’-
CGACGTTGTAAAACGACGGCCAGTTTTTTTTTTT'ITTTT -3’) that consisted of
M13-20 sequence primer and oligo (dT)16 (Tao et al. 1999). Pru-T2 primer (5’-
TS'ITSTTGSTTTTGCTTTCTTC -3’) (Tao et al. 1999) derived from the cDNA
sequence corresponding to the signal peptide sequence of S-RNases of sweet cherry was
used in 3’ rapid amplification of cDNA ends (3’ RACE) with M13-20 primer as the
adapter primer. PCR was performed using a program of 30 cycles at 94 °C for 30 sec, 56
°C for 30 sec, and 72 °C for 1 min with an initial denaturing of 94 °C for 3 min and a
final extension of 72 °C for 7 min. The PCR reaction mixture contained 10 mM Tris-HCl

(pH 8.3), 50 mM KCl, 1.5 mM MgC12, 200 11M each of dNTPs, 400 nM each of primers,

51

template cDNA equivalent to the amount synthesized from 0.1 ug of the total RNA, and
1 U of Ex Taq polymerase (TaKaRa Shuzo Co, Shiga, Japan) in a 50 uL reaction volume.
The PCR products were subcloned into the T-A cloning vector (pGEM-T Easy Vector
System; Promega, Madison, Wisc.). DNA was sequenced using the Dye Terminator
Cycle Sequencing Kit (Applied Biosystems, Tokyo, Japan) and the ABI PRISMTM 310
Genetic Analyzer (Applied Biosystems, Tokyo, Japan). The deduced amino acid
sequences of four different cDNAs from ‘RS’ obtained in this study and two different
cDNAs from ‘EB’ (Yamane et al. 2001) were aligned using Clustal X (Thompson et a1.

1997)

DNA isolation and Southern analyses from the parents and progeny in the linkage
mapping population

Young, unfolded leaves were collected from the parents and the progeny, placed
on dry ice, stored at -80 C overnight and then lyophilized for 48 h. DNA was isolated
using the CTAB method described by Stockinger et al. (1996). ‘EB’, ‘RS’, and the
linkage mapping progeny were evaluated using Southern blotting following HindIII
digest which had previously been demonstrated to differentiate all the ‘EB’ and ‘RS’ S-
RNases (Yamane et al. 2001).

Six pg of DNA for both parents and 85 progeny was digested with HindIII
(Boehringer Mannheim Biochemicals, Indianapolis), run on a 0.9 % agarose gel for 36 h
at 30 V, and transferred to a nylon membrane (Hybond-N+, Amersham) according to
Wang et al. (1998). PCR amplified fragments of the S6-RNase cDNA from sweet cherry

(Tao et al., 1999) were used as the probe. Probes were radiolabelled with 32P-dCTP

52

(DuPont, Boston) using the random primer hexamer-priming method described by
Feinberg and Vogelstein (1983). After hybridization at 60°C for 16 hours and high
stringency washes (2 X 30 min with 2 X SSC and 1 % SDS followed by 2 x 30 min with

0.2 X SSC and 0.5 % SDS at 60 °C), radioactive signal was detected on X-ray films.

Inheritance and linkage analysis

Segregation of the S-alleles that were present in one parent but absent in the other
parent, were tested for their fit to the expected 1:1 (presencezabsence) ratio. So which
was present in both parents was tested for its fit to a 3 :1 (presencezabsence) ratio.

The most informative markers for linkage mapping from a pseudo-testcross
mapping population are single dose restriction fragments (SDRF) that differ between
both parents and segregate l : 1 (presence : absence) (Wu et al. 1992). Therefore, S-
alleles which differed between both parents and fit a 1:1 ratio at the 5% level were
combined with the existing marker segregation data previously used to construct the ‘RS’
x ‘EB’ linkage map (Wang et al. 1998).

Linkage analysis was done with JoinMap V2.0 (Stam 1993) using a minimum

LOD score of 3 .0. Distances are presented in centi-Morgans calculated by the Kosambi

function.

53

Results

Pollen tube growth studies of sour cherry and sweet cherry interspecific crosses
‘Sato Nishiki’ (S 3S6) pollen was able to grow the full length of both ‘Crisana’
(S 1S4Sd) and ‘Tschernokorka’ (SgSaSc) styles (Table 3.1). ‘Rainier’ (S 1S4) pollen was able
to grow the full length of the ‘Tschernokorka’ styles (Table 3.1). However, ‘Rainier’
pollen tube growth was arrested half way down the ‘Crisana’ styles (Table 3.1) and
swelling was observed at the pollen tube tips. This suggests that the S 2- and S4-RNases
from ‘Crisana’ were able to recognize and inhibit the S 1- and Sr-pollen from ‘Rainier’.
‘Tschernokorka’ pollen grew the full length of both ‘Rainier’ and ‘Schmidt’
(S2S4) styles (Table 3.2). ‘Crisana’ pollen was also able to grow the full length of
‘Schmidt’ styles; however, ‘Crisana’ pollen was arrested halfway down the ‘Rainier’
styles (Table 3.2). This suggests that the S 2- and S4-RNases from ‘Rainier’ were able to
recognize and degrade the 2x pollen tubes from ‘Crisana’ that would either be

heteroallelic or homoallelic for the S 1-, S4- and/or Sd-pollen S-alleles.

cDNA cloning from ‘RS’

3’ RACE cDNA clones encoding four S-RNases that had previously been
determined to be in ‘RS’, S0, S1,, Sc and S6, were cloned and sequenced. Two of the three
unique S-RNases fiom ‘EB’, S0 and S4, had previously been cloned and sequenced
(Yamane et al. 2001). A cDNA for the S6,", the third S-allele postulated to be in ‘EB’,
could not be identified (Yamane et al. 2001). However, this allele was suspected to be

functionally similar to the S6-allele due to complete identity between the S6 sweet cherry

54

Table 3.1 Cross-compatibility results for pollination of sour cherry styles with
sweet cherry pollen based on examination of pollen tube growth in

styles 72 hours after pollination

 

 

Style parent Pollen parent (S-allele genotype)
(S-RNases)’ ‘Rainier’ (S 1S4) ‘Sato Nishiki’ (S 3S6)

‘Crisana’ (S 1S4Sd) Incompatible Compatible

‘Tschernokorka’ (SgSaSc) Compatible Compatible

 

’ Three different S-RNases have been identified in each parent. At this time, it is not
known which of the three S-RNases is present in two COpies.

Table 3.2 Cross-compatibility results for pollination of sweet cherry cultivars
with sour cherry pollen based on examination of pollen tube growth in

styles 72 hours after pollination

 

 

 

Style parent Pollen parent (S-RNases)a
(S-allele genotype) ‘Crisana’ (S 2S4Sd) ‘Tschernokorka’ (SgSaSc)
‘Rainier’ (S2S4) Incompatible Compatible
‘Schmidt’ (S2S4) Compatible Compatible

 

‘ Three different S-RNases have been identified in each parent. At this time, it is not

known which of the three S-RNases is present in two copies

55

sequence and the sequence amplified from ‘EB’ using the Pru-C2 and PCE-R primers
that span the two hyper-variable regions present in the Prunus S-RNases (Tao et al.
1999; Yamane et al. 2001). The amino acid alignment of the four S-RNases from ‘RS’
with the two S-RNases from ‘EB’ is presented along with the amino acid sequences of
the sweet cherry S4 and S6 sequences (Genbank sequence accessions AB028154 and
ABOlO305, respectively) (Figure 3.1). The partial amino acid sequence of the S,-
RNase from ‘RS’ was identical to the sequence of the Sa-RNase from ‘EB’ determined
previously (Y amane et al., 2001) (Figure 3.1). The deduced amino acid sequences from
the Sb- and Sc-cDNA clones contained the two active domains shared by other T2/S-
RNases and the five regions that are conserved among rosaceous S-RNases. In addition,
seven cysteine residues and an N-glycosylation site conserved among other rosaceous
S-RNases were present in the amino acid sequences of the Sb- and Sc-RNases. However,
the sequences of the cDNAs encoding the Sb- and Sc-RNases were not identical to the
DNA sequences of any known S-RNases, suggesting that they encode novel S-RNases.
The novel S2,- and Sc-RNases share 63 to 80 % identity with other sweet cherry S-
RNases, within the range of amino acid sequence identity observed among Prunus S-
RNases. These results indicated that four different kinds of S-RNases, corresponding to
the four S-alleles in the genome of ‘RS’, are present in the style of ‘RS’.

Inheritance and linkage analysis of the S-RNases in the ‘RS’ x ‘EB’ mapping
population

The S-RNase RFLP profiles following HindIH digestion for ‘RS’ and ‘EB’ agreed
with the previous report (Yamane et al. 2001). ‘RS’ exhibited four fragments of 6.4 kb,
5.8 kb, 5.1 kb, and 4.6 kb which correspond to the S-RNases, Sq, S6, S2, and Sc,
respectively (Figure 3.2). The four fragments exhibited by ‘EB’ correspond to the three
S-RNases S6", (9 kb), S, (6.4 kb) and S4 (6.1 kb and 5.6 kb) (Figure 3.2). Partial genomic

sequences of the ‘RS’ and ‘EB’ derived S6- and San-RNases, respectively, were identical

56

Figure 3.1 Amino acid sequence alignment of four S-RNases from ‘RS’, Sa, Sb, Sc and
S6, two from ‘EB’, S0 and S4, (Yamane et al. 2001) and the sweet cherry S4- and S6-
RNases. The alignment was generated by CLUSTAL X (Thompson et a1. 1997). Gaps are
marked by dashes. The five conserved regions, C1, C2, C3, RC4 and C5 (U shijima et al.
1998) are marked with solid boxes, and the hypervariable region, RHV (U shijima et al.
1998), reported in rosaceous S-RNases is marked with a dotted box. Conserved amino

acid residues are designated by asterisks under the sequences.

57

 

PC-Sb-RS
PC-Sc-RS
Pa-SG-RS
PC-Sa-RS
PC-Sa—EB
Pa-S4-EB

PA-SS
PA-S4

PC-Sb—RS
PC—Sc-RS
PL-SS-RS
PC-Sa-RS
PC-Sa-EB
PA-S4-EB

Pl-SS
PA-S4

PC-Sb—RS
PC-Sc—RS
PA-SG-RS
PC-Sa—RS
PC-Sa-EB
PA-S4-EB

Pl-SS
PA-S4

PC-Sb-RS
PC-Sc-RS
PA-SG—RS
PC-Sa-RS
PC-Sa-EB
PA—SQ—EB

PA-SG
PA-S4

PC-Sb-Rs
PC-Sc-RS
PL-SB—Rs
PC-Sa-RS
PC-Sa-EB
PA-SQ—EB

PA-SS
PA-S4

(31

 

1 __________________ rcrxnsrsrdsrvrrorvoovpTTTCILRKK-
1 __________________ Lcrrnsr--GSYVYFQFVQQUPPTTCRLSSK-
1 ------------------- LCFIHSNF—GSYVYFQFVQQUPPTNCRVRIKR
1 __________________ LcrrnerDGSYDYFQFVQQUPPATCSLSRT-

1 HVTLKSSLAFLVLAFALFLCFIHSTGDGSYDYFQFVQQUPPATCSLSRT-
1 HAILKSTLLFLVLAFAFFICYVHSS--GSYDYFQFVQQUPPTNCRVRNK-

1 HAHLKSSPLFLVLAFLFFLCFIHSN--GSYVYFQFVQQUPPTNCRVRIKR
1 HAILKSTLAFLVLAFAFFICYVHSS--GSYDYFQFVQQUPPTNCRVRNK-

* it it: tittttttt *

C2 “"3113." .....
51 -CSQPRPLQI| 'S YSNPTRPSNCIGiQFNFTKVYPHHRflKLKR
51 PSHQHRPFQ" '-NYSNPRKPSNCNCSQFDDRKVYPDLRSPLKR
51 PCSSPRPLQ FTIHGLUP‘NYSNPRHPSNCTGPDF-KRILSPQLRSKLQT
51 PCYKPRPPQI'TIHGLUP~NYSNPKRPSNCRGSFFDSRKVYPQLRLNLKI
51 PCYKPRPPQ'ITIHGLU'~NYSNPKRPSNCRGSEFDSRKVYPQLRLNLKI
51 PCTKPRPLQ FTIHGL"= YSNPRHPSKCTGSLFNFRKVYPQLRSPLKI

' r

 

 

 

 
 
   
 
 
   
 
   
  
  
  
 
 

' r
51 PCSSPRPLQIITIHGL"~ SNPRHPSNCTGfioF-KRILSPQLRSKLQT
51 PCTKPRPLE VTIHGL"4NYSNPRHPSKCTGSEENIBEEXEgkfigbLKI

it 8 titttttttttttt it i t t t t t

(33

101 AUPDVESGND
101 SUPDVEGGND
101 SUPDVESGND
101 SUPNVKSGND
101 SUPNVKSGND
101 SUPDVESGND

AHURS
QTLNQFQYFERSHDHWHS
AHUHS
QTLNQHQYFERSDEHUNS
EHUNS
IS

101 SUPDVESGND
101 SUPDVESGND

it t

AHUHS
IS

tttt tit tttttt ft it t *3 I

fit tttttf

   
 
 
 
 
 
 
   
 
    

151
151 LKNASIV
151 LKNLSIV
151 LKKAQIV
151 LKKAQIV
151 LKNLSIV

ATQKUSYSDIVAPIKAATKRTPLLRCKQD ____________
AKQRUKYSDIVSPIKGATGRTPLLRCKRDPA ----------
PTQTUKYSDIVAPIKALTKRTPLLRCKQD ____________
ATRTUKYSDILSPIKAATNTTPILRCKPDPAQSKSQPSQPK
ATRTUKYSDILSPIKAATNTTPILRCKPDPAQSKSQPSQPK
ATKNUTYSDIVSPIKRATKRTPLLRCKYD ____________

151
151

TQTUKYSDIVAPIKAATKRTPLLRCKQD ............
ATKNUTYSDIVSPIKRATKRTPLLRCKYD ____________

t ttti fit it t 31*? t

(35

QLLHLHEVVFC EYNALKQIDCNRTSACGNQQTISFQ
LLH--EVVFC YNALKQIDCNRTAGCKNQRAISFQ
LH—-EVVFCYEYNALKQIDCNRTSGCQNQPAISFQ
QLLH--EVVFCYDYHAKKQIDCNRT-GCLN-KDISFQ
QLLH--EVVFCYDYHLKKQIDCNRT-GCLN-KDISFQ
QLLH--EVVFCYEYDALKQIDCNGTAGCPNQKVISFQ

201 --KK
201 --TN
201 --KN
201 SPQK
201 SPQK
201 --KS

201 --KN
201 --KS

YEYNALKQIDCNRTSGCQNQPAISFQ
YEYDALKQIDCNGTAGCPNQKVISFQ

it? tttttt * t titttt t t 3 fit?

 

58

 

50
50
50
50
50
50

50
50

100
100
100
100
100
100

100
100

150
150
150
150
150
150

150
150

200
200
200
200
200
200

200
200

242
242
242
242
242
242

242
242

 

RSEB

Figure 3.2 Genomic blot analysis of ‘RS’, ‘EB’ and eleven progeny. Six micrograms of
genomic DNA were digested by HindIII and hybridized to the cDNA encoding the S6-

RNase.

59

suggesting that they are the same S-RNases and that the RFLP polymorphism occurs
outside the S-RNase coding region (Yamane et al. 2001).

S-RNase segregation in 85 progeny fiom the ‘RS’ x ‘EB’ mapping population was
determined (Table 3.3). Segregation of the S-alleles that were present in the maternal
parent, ‘RS’, but absent in the paternal parent, ‘EB’, (Sb, Sc, and S6) fit the expected 1:1
ratio (Table 3.3, Figure 3.2). Segregation of the Sa-allele, which was present in both ‘RS’
and ‘EB’, fit a 3:1 ratio, which was expected if the S-RNase was present in only one dose
in each parent and there was no pollen selection. Segregation for the S4-allele which was
present in ‘EB’ and absent in ‘RS’ did not fit a 1:1 ratio which would be expected if the
S4-allele was only present in a single dose. However, segregation of S4 in ‘EB’ fit a 5:1
ratio suggesting that there are two S4-alleles exhibiting tetrasomic inheritance and
therefore the ‘EB’ S-RNase genotype is presumed to be ngS4SrSa. The S6", in ‘EB’ that
could be distinguished from the Sg-allele in ‘RS’ by RFLP analysis following HindIII
digest (Figure 3.2) was not present in any of the progeny.

The three S-RNase alleles that fit a 1:1 segregation ratio, Sb, Sc and S6, all from
the maternal parent, RS, were used for linkage analysis. The Sb-allele mapped to ‘RS’
linkage group 6 of the framework map constructed by Wang et a1. (1998) (Figure 3.3).
The ‘RS’ linkage group 6 consisted of 17 markers spanning 34.4 cM and the Sb-locus
mapped 4.5 cM from the marker placed at one end of this linkage group. The other two S-
alleles, S6 and Sc, were linked to each other at a distance of 23.2 cM and unlinked to any
other previously identified markers. When the progeny SI and SC phenotypes were

entered as data in the linkage map analysis, the SI and SC phenotypes did not segregate

60

 

Table 3.3 RFLP segregation of S-RNase alleles in the ‘RS’ x ‘EB’ mapping

 

 

population I
S-allele RS EB Expected Presence: absence x2 value

ratio ratio in progeny y

S, + + 3 :1 69 : 15 2.29

Sb + - 1 :1 34 : 51 3.4

Sc + - 1:1 46 : 37 0.98

S; - + 1:1 77: 8 56.0 **
5:1" 3.25

S6 + - 1:1 45 : 40 0.29

S6", - + 1:1 0 : 85 85.0 **

 

** denotes significance at the 0.001 level

2 The S-genotypes of ‘RS’ and ‘EB’ were provisionally determined to be

SaS’bSoSg and SaS4S6m, respectively, by Yamane et al. (2001)

y Only the data for those progeny that could be unambiguously scored for

each S-allele was included

" 5:1 is the expected segregation ratio for an allele that is present in two

copies which exhibits tetrasomic inheritance

61

j— EFllD

-r- Sb

45

4.2

---- B46101:
3.2

15
21]

---— EFlSZb
CPM104

CPMfl9a

 

 

3.6
m— CPMZJC

'"r- EFl62a

1.?

2.3
2.5

"" EF1761
--— CPMZBd

7.2

-'- EF176j

1'?-[-CPMZJd

 

 

Figure 3.3 Genetic map for the sour cherry linkage group 6 obtained with the ‘RS’ x
‘EB’ mapping population showing the location of the Sb-RNases (Sb). The framework
map was created by Wang et al. (1998). Markers shown on the right are identified by the

probe followed by a letter (i.e., a, b, c, etc.) when more than one marker is generated

from a single probe.

62

with any markers suggesting that SI and SC in sour cherry is controlled by segregation at

more than one locus.

SI and SC phenotypes of progeny from the ‘RS’ x ‘EB’ mapping population

Both self- and outcross-pollen grew the firll length of ‘RS’ and ‘EB’ styles during
pollen tube grth studies, confirming that both cultivars are SC. The S1 or SC
phenotype for 65 of the 85 individuals in the mapping population could also be
determined. Thirty-nine progeny were determined to be SC, while 26 were SI. Nine
other trees had died before their phenotype could be determined. Six trees produced no
functional pollen during either of the two pollination seasons, and were thus concluded to
be male sterile. Low fertility among the remaining six progeny caused ambiguous S1 or
SC phenotype results, thus no phenotype could be assigned.

When the SI and SC phenotypes were compared with the RFLP S-RNase patterns
detected for the 65 progeny tested for both traits, the presence/absence of one or more S-
RNase fragment(s) was not associated with either S1 or SC. In addition, certain S-allele

genotypes segregated for SI and SC phenotype.

Discussion

Inter-specific crosses between sweet and sour cherry demonstrate that gametophytic self-
incompatibility exists in sour cherry and that the recognition of common S-alleles has

been maintained in spite of polyploidization. The results from the reciprocal crosses of

63

‘Crisana’ (S1S4Sd) and ‘Rainier’ (S 2 S4) indicate that the S1- and S4-stylar and pollen
components in the two selections are functionally similar.

The incompatible reaction in the cross ‘Crisana’ (S 1S4Sd) x ‘Rainier’ (S 1S4) can be
explained by the recognition of the ‘Rainier’ S 1- and S4-pollen S-genes by the ‘Crisana’

S 2- and S4-RNases, respectively. However, an explanation for the incompatibility of the
reciprocal cross, ‘Rainier’ x ‘Crisana’, is less straightforward. Since it is expected that
like most sour cherry cultivars, ‘Crisana’ exhibits occasional inter-genomic pairing
(Murawski and Endlich 1962) six types of pollen would be possible: heteroallelic (i.e.
S2S4, SISd, S4Sd) and homoallelic (S 1S 2, SS, Sde) depending on which allele is in double
dose. The relative frequencies of these pollen types would also be dependent on dosage
and pairing configuration. However, without confirming the precise pollen genotypes
and the firnctional activity of the different S-haplotypes it is premature to speculate on the
mechanism of pollen recognition and inhibition.

The results from the cross ‘RS’ x ‘EB’ suggest that only one S-allele match
between the style and pollen is necessary to render diploid pollen incompatible. In the
cross, the genotypes of ‘RS’ and ‘EB’ were determined to be SaSbScSC; and SaS4S4S6m,
respectively. In the progeny, it was possible to follow the inheritance of the ‘RS’-derived
S6-allele and the ‘EB’-derived San-allele because these two alleles could be differentiated
by RFLP analysis following HindIH digestion. Since ‘EB’ only has one S6m-allele, all the
pollen containing S6", are expected to be heteroallelic. However, in this cross no progeny
individuals contained the ‘EB’-derived San-allele. This observation is in contrast to
results from several Solanaceous species where heteroallelic pollen loses its SI phenotype

(Livermore and Johnstone 1940; Stout and Chandler 1942; Crane and Lewis 1942;

64

Brewbaker 1954; Pandy 1968; de Nettancourt et al. 1974; Chawla et al. 1997; Entani et
al. 1999; Golz et al. 1999; Luu et al. 2001)

The previously reported inability to identify any Sg-RNase associated protein or
cDNA in ‘EB’ (Yamane et al. 2001) suggests that the San-RNase is not firnctionally
active in the ‘EB’ style. The segregation data suggests however, that the ‘EB’ derived
San-pollen component is firnctional and is inhibited by the ‘RS’ S6-RNase. It would
therefore be postulated that in the reciprocal cross, ‘EB’ x ‘RS’, both the ‘RS’- and ‘EB’-
derived S6-alleles should be inherited by a portion of the progeny.

Given the putative S-genotype of ‘EB’ (SaS4S4S6m) and the inability of pollen
containing the San-allele to successfully fertilize ‘RS’, all progeny should inherit at least
one copy of the S4-allele fi'om the ‘EB’ parent. However, eight of the 85 progeny that
were genotyped for their S-RNase alleles did not contain an S4-RNase. One possible
explanation is that the San-allele region of ‘EB’ that contains the pollen S and S-RNase
genes has been translocated leaving an S-null allele. Therefore, these eight progeny that
do not contain an S4-RNase would have obtained Sa- and S-null alleles from the ‘EB’
parent. These plants consisted of both SC and SI types.

Both linkage mapping parents, ‘RS’ and ‘EB’, have one Sa-RNase allele. Progeny
segregation for this allele (3: 1, presentzabsent) suggested that both parental copies of the
Sa-allele were inherited by the progeny. This result would be expected if heteroallelic
pollen from ‘EB’ carrying the Sa-allele and another S-allele were SC. However, data
from the progeny did not support this hypothesis. Sixteen progeny were obtained that,
like both parents, contained only one copy of the Sa-allele. Since five of these 16

progeny were SI, heteroallelic pollen with just one S0 is not sufficient by itself to cause

65

the plant to be SC. In the sour cherry cross, the ability of pollen with an Sa-allele to grow
down a style containing an Sa-RNase may be possible if at least one of the Sa-alleles is
non-functional due to a mutation in either the S-RNase or pollen S-gene coupled with the
presence of a second non-functional S-allele in either the style or pollen.

Whereas the current study provided additional evidence of S-RNase segregation in
sour cherry, the actual cause of SC versus S1 in sour cherry was not determined. For
example, no S-RNase(s) have been found to co-segregate with SC in the ‘RS’ x ‘EB’
population, as has been found in some SC mutants (Tao et al. 2000), so it is unlikely that
a single S-RNase mutation is responsible for causing self-compatibility. One possible
mechanism for the existence of SC in sour cherry is the existence of mutations in the S-
RNase or pollen S-gene of a number of S-alleles. Therefore the identification of the
pollen S-gene(s) in sour cherry is likely to be crucial to the understanding of SC and 81 in
sour cherry. In addition, certain progeny with similar S-haplotypes differed with respect
to their SI or SC phenotypes. This suggests that modifier genes may have a role in
modulating the interaction between the S-pollen protein and the S-RNase. Modifier
genes have been demonstrated to be required for normal SI firnction in Nicotiana
(McClure et al. 1999).

Along with the crossing studies described above, amino acid matches of the sweet
cherry and sour cherry S4- and S6-alleles, indicate that the alleles that were suspected to
be in common between these two species are identical. This is additional evidence that
gametophytic SI occurs in sour cherry and that it is regulated by a similar mechanism as
found in other gametophytic SI species in which a stylar S-RNase interacts with a pollen-

specific molecule to regulate the SI or SC response.

66

Since sour cherry is an allotetraploid, it is likely that the two S-loci would map to
homoeologous linkage groups. The Sb-RNase mapped to the expected position on Prunus
linkage group 6 indicating that this linkage relationship is maintained among sour cherry,
sweet cherry and almond. The two other S-RNases, Sc and S6, mapped together on what
might represent a new homoeologous linkage group 6 that had previously been
undetected by Wang et al. (1998) due to low marker density on the sour cherry map.
However, Sc and S6 would have been expected to map to the same location and it is not
clear why they are separated by over 20 cM. Selection for or against certain S-alleles or
other alleles in the S-locus region could have occurred. In addition meiotic irregularities
prevalent in sour cherry can complicate linkage analysis. Although sour cherry is an
allotetraploid predominately exhibiting disomic inheritance, it also exhibits tetrasomic
inheritance (Beaver et al. 1993) and quadrivalent pairing characteristic of an
autotetraploid (Wang 1998). For example, all twenty of the ‘EB’ metaphase I pollen
mother cells (PMC) examined had some non-bivalent pairing with at least one
quadrivalent per PMC. Therefore the tetrasomic inheritance exhibited by the ‘EB’ S4-
alleles is not unexpected. However, it is also possible that the Sc —S6 linkage result is
caused by an actual change in the physical location of the S-locus. This is not
unprecedented since a translocation has been confirmed in almond between Prunus

linkage groups 4 and 6 (P. Arus, pers. comm).

67

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72

CHAPTER 4

GENETIC CONTROL OF SELF-INCOMPATIBILITY AND
SELF-COMPATIBILITY IN TETRAPLOID SOUR CHERRY
(PR UNUs CERASUS L.)

73

Abstract

Gametophytic self-incompatibility (S1) in diploid species generally breaks down due to
polyploidy, resulting in self-compatible (SC) tetraploid species. The diploid sweet cherry
and the tetraploid sour cherry represent an exception, as sour cherry individuals can be
either S1 or SC. To investigate the genetic control of SI and SC in sour cherry, the
segregation of S-haplotypes in a total of 794 progeny from six sour cherry self-
populations and 15 inter-specific crosses between sweet and sour cherry involving six SC
sour cherry selections were analyzed. Results indicate that the breakdown of S1 in
Prunus is caused by the accumulation of non-functional S-haplotypes that are incapable
of S-haplotype-specific rejection of pollen, rather than due to the competition between
pollen-S products in heteroallelic pollen, as commonly observed in the Solanaceae. In
total, four firlly firnctional S-haplotypes and six non-functional S-haplotypes were
discovered in these six sour cherry selections. A hypothesis regarding the genetic control
of SI and SC in sour cherry was developed and verified in self-pollination and crossing
experiments. The implications of these findings on sour cherry breeding and on our

knowledge of gametophytic SI are discussed.

74

Introduction

Gametophytic self-incompatibility (GSI) is a common mechanism for promoting
out-crossing in flowering plants (de Nettancourt, 1977). In GSI, self-incompatibility (S1)
is determined by a single, multi-allelic locus, called the S-locus, in which the
compatibility of a cross is determined by the haploid genome of the pollen and the
diploid genome of the pistil. Pollen tube grth is arrested if the pollen tube has an S-
allele in common with one of the two S-alleles in the style. The S—locus contains a
minimum of two genes, one controlling the style-specificity and the other the pollen-
specificity of the SI reaction. The stylar-determinant is an RNase (S-RNase) (Anderson,
1986; McClure et al., 1989), which is specifically expressed in the pistil and specifically
degrades rRNA of incompatible pollen (McClure et al., 1990). The identity of the pollen-
S gene remains unconfirmed; however, S-locus linked F-box genes have been proposed to
be the pollen-S gene in Anthirrinum (SLF; Lai et al., 2002), Prunus mume (SLF; Entani et
al. 2003) and P. dulcis (SFB; Ushijima et al. 2003). The inability to transform these
species precludes the necessary transformation experiments to verify that one of these F-
box genes is the pollen-S gene. Transformation experiments involving a recently
discovered S-locus F-box gene in Petunia inflata (PiSLF) suggests that it is the pollen-S
gene in the Solanaceae (Sijacic et al., 2004).

The S-RNase gene from the Solanaceae and the Rosaceae are thought to be
orthologous (Igic and Kohn, 2001). However, several differences between Prunus and
other Rosaceous and Solanaceous species have been reported. The S-RNase gene of

Prunus contains two introns, whereas the S-RNase for all other Rosaceous and

75

Solanaceous species contains only one (Igic and Kohn, 2001). Second, the putative
pollen-S F-box genes isolated from Antirrhinum (Lai et al., 2002) and Prunus (Entani et
al., 2003; Ushijima et al., 2003) do not appear to be orthologous. Third, screens for
pollen-part mutants (PPMs) in the Solanaceae have failed to reveal a PPM caused by the
mutation of the pollen-S gene (Pandey 1967; van Gastel and de Nettancourt, 1975; Golz
et al., 1999; Golz et al., 2000). Instead, all PPMs have included duplications of the
pollen-S gene, suggesting that a deletion or knock out of the pollen-S gene is lethal.
However, two PPMs, S; from sweet cherry and Sf from P. mume, that were caused by the
knock out of pollen-S function have been characterized in Prunus (U shijima et al., 2004).
Together, these findings suggest that the identity and/or firnction of the pollen-S gene
may differ in Prunus and the Solanaceae.

GSI typically breaks down as a result of polyploidy (Crane and Lawrence, 1931;
Crane and Thomas, 1939; Livermore and Johnstone, 1940; Stout and Chandler, 1941;
Pandey 1968; de Nettancourt et al., 1974). It is hypothesized that the competition
between multiple different pollen-S products in a single pollen grain eliminates the ability
of the pollen to initiate an SI reaction, allowing these heteroallelic pollen grains to
successfully grow through styles despite the presence of their cognate S-RNases (Stout
and Chandler, 1942; Lewis 1943; Chawla et al., 1997; Entani et al., 1999). However, a
recent comparison of ploidy level and the presence of self-compatibility (SC) among
angiosperrns indicates that there is no association between ploidy level and SC (Mable

2004). The frequency of SI and SC species was not significantly different for diploids

and tetraploid species.

76

Sweet cherry (P. avium) is a diploid species that has a classical RNase-based GSI
system (Boskovic and Tobutt, 1996) with 17 identified S-haplotypes (Mathews and Dow,
1969; Tao et al., 1999; Boskovic and Tobutt, 2001; Hauck et al., 2001; Sonneveld et al.,
2001; Wiersma et al., 2001; Choi et al., 2002; Sonneveld et al., 2003; Iezzoni et al., 200x;
Wunsch and Horrnaza, 2004). Sweet cherry is one of the progenitor species of the
allotetraploid sour cherry (P. cerasus). Sour cherry also contains a GSI system
characterized by the premature cessation of incompatible pollen tube growth in the styles
(Lansari and Iezzoni, 1990; Yamane et al., 2001). In addition, reciprocal inter-specific
crosses between sweet and sour cherry indicate that sour cherry has retained its ability to
reject pollen in an S-haplotype-specific manner (Hauck et al., 2002). However, a partial
breakdown of SI has occurred in sour cherry, resulting in the existence of both SC and S1
types (Lech and Tylus, 1983; Redalen, 1984; Lansari and Iezzoni, 1990). The genetic
cause of the partial breakdown of S1 in sour cherry is unknown.

It remains to be seen whether sour cherry pollen that contains two different
pollen-S products loses its SI phenotype, as in the Solanaceae, or whether a different
mechanism causes the observed partial breakdown of SI in sour cherry. A second
possible mechanism for the breakdown of S1 is that mutations in a modifier gene, such as
the HT gene from Nicotiana (McClure et al., 1999), causes the breakdown of S1 in sour
cherry, as suggested by progeny from a cross between Rheinische Schattenmorelle (RS)
and Erdi B6term6 (EB) that contain the same S-RNase phenotype but segregate for SI and
SC (Hauck et al., 2002). A third possibility is that mutations in one or both of the
specificity components, S-RNase or pollen-S, have accumulated and the inability of these

non-firnctional S-haplotypes to carry out S-haplotype specific rejection causes the partial

77

breakdown of S1 in sour cherry. One such non-functional sour cherry S-haplotype, S6,",
has been characterized and found to have a firlly functional pollen-S gene that was
identical to that of the S6 haplotype of sweet cherry. The S-RNase of the San-haplotype,
however, was non-firnctional due to the insertion of a 2.6 kb fragment upstream from the
Sg-RNase gene, which eliminated gene expression (Yamane et al., 2003). A more in
depth genetic characterization of S-haplotypes is necessary to differentiate between these
three possibilities.

The ultimate goal of this research was to determine the genetic control of SI and
SC in sour cherry. In this study, systematic genetic analyses of previously defined S-
haplotypes were conducted using both inter-specific crosses between sweet and sour
cherry and sour cherry self-populations. Finally, a hypothesis was developed for the
genetic control of SI and SC in sour cherry and verified using self-pollination and
crossing experiments. Implications of the findings on the evolution and the effects of

polyploidy on GSI are discussed.

Materials and Methods

Plant material

All sweet and sour cherry cultivars, along with their proposed S-haplotypes, used
in this study are listed in Table 4.1. The sour cherry S-haplotype nomenclature proposed
by Yamane et al. (2001) is used in this paper, with the S4, S6 and Sta-haplotypes

representing RFLP profiles identical to S-haplotypes in sweet cherry, and Sa, Sb, S4, and

78

Table 4.1: Sweet and sour cherry cultivars used in this study, with their proposed S-

 

 

haplotypes
Cultivar 81 or SC S-haplotype‘
Sweet Cherry
Chelan SI S 3S9
Emperor Francis (EF) SI S 3S4
Gold . SI S3S6
Regina SI S 1S 3
Schmidt SI S 254
Sour Cherry
Cigany 59 SC SaSg/SaSb
Erdi B6term6 (EB) sc Srs6/Sasx "
Montmorency SC 56.313'/S0Sx b"
Rheinische Schattenmorelle (RS) SC S6SI3'/ SaSb ‘
Surefire SC S4513/ San b"
Ujfehértoi fi'utds (UF) SC s.s./ Sds, "

 

‘ Sour cherry is an allotetraploid. The homologous pairing of S-haplotypes are
designated. Homologous pairing was determined based on observed segregation of S-
haplotypes in this study.

b In those sour cherry cultivars where only three S-haplotypes could be identified, 5,, is
used to denote either a null allele or the double dosage of one of the other S-haplotypes.

cS 2 3' was previously named Sc (Yamane et al., 2001). Alignment of the predicted amino
acid sequence of the S13-RNase from sweet cherry (GenBank accession number:

A] 63 5276) and Sc from sour cherry (Hauck et al., 2002) indicates that these two S-

RNases are identical.

79

Se having unique RFLP banding profiles. The previously reported Sc has been renamed
S13' due to identical amino acid sequences of the sweet cherry S13-RNase (GenBank
accession number: A] 63 5276) and the Sc-RNase (Hauck et al., 2002). The “"’ indicates a
hypothesized pollen-part mutation. The trees are located either at the Michigan State
University Clarksville Horticultural Experimental Station (CHES), Clarksville, Mich. or

the MSU Northwest Research Station (NWRS), Traverse City, Mich.

Field crosses and self-pollinations

The inter-specific crosses and self-pollinations that were analyzed in this study are listed
in Tables 4.2 and 4.3, respectively. Anthers were collected from flowers in the late
balloon stage, allowed to dry at room temperature overnight, and either used immediately
or stored in glass vials at -20°C with a desiccant. One day prior to bloom, about 200
flowers per cross were emasculated. The next morning, the dried anthers were crushed
with glass rods and applied directly to the stigmas. Immediately following fruit ripening,
the seed were harvested, cleaned from the surrounding fruit, and stored at -20°C until

used for DNA isolation.

DNA isolation

DNA isolation fiom leaves of parents: Young, unfolded leaves of firlly-grown trees were
placed on dry ice immediately following collection, stored at -80°C overnight and then

lyophilized for 48 h. DNA isolation was performed using the CTAB method described

by Stockinger et al. (1996).

DNA isolation fiom seed: Seed fiom the crosses were harvested shortly after ripening
and stored at -20°C. The testa was removed to allow DNA extraction from the embryo

and cotyledons. The embryo and cotyledons were crushed using liquid nitrogen and

80

Table 4.2: Reciprocal inter-specific crosses between sour cherry and sweet cherry used

to investigate the functionality of the S4, S6 and Sg—haplotypes from sour

 

 

 

 

 

cherry.
Parents used in cross S-haplotype No. of grogeny analyzed

Sour cherry Sweet cherry tested sour x sweet sweet x sour
Cigany 59 (SdoSQSaSb) Gold (S 3S6) S6 36 40
Cigany 59 (SchcS'aSb) Chelan (S 3S9) S9 7 0 ‘

EB (S4S6mSan) BF (S354) S4 10 20

EB (S4S6mSan) Gold (S3S6) S6 33 14
Montmorency (S6Sj3'San) Gold (S 3S6) S6 55 15

RS (S6SI3'SaSb) Gold (S 3S6) S6 31 13
Surefire (S4S23'S0Sx) EF (S 3S4) S4 30 3 7

UP (5.519.539 Schmidt (525.) s. 45 o "
UF (5.3.3.5.) BF (S354) S. o b 36

 

" Chelan was not used as the maternal parent. Thus, the functionality of the pollen-S 9

gene from Cigany 59 could not be tested.

b Different sweet cherry testers were used for the reciprocal crosses with UF.

8]

Table 4.3: Sour cherry self-populations analyzed to determine the

functionality of the 513', S2,, S2,, S2 and Se-haplotypes.

 

 

 

Cultivar self-pollinated No. of progeny analyzed
Cigany 59 37

Erdi B6term6 8
Montmorency 135
Rheinische Schattenmorelle 54

Surefire 36

Ujfehértoi firrtos 102

 

82

mixed in a buffer consisting of 150 mM Tris-HCl (pH 8.0), 20 mM EDTA, 800 mM
NaCl, 0.25% SDS, 1% B-mercaptoethanol and 1% CTAB. The DNA was purified by

two chloroform extractions and precipitated using isopropanol. See Appendix A for

complete protocol.

PCR amplification of S-RNases

The primer combination, Pru-C2 and PCE-R which hybridized to conserved
regions flanking the second highly variable intron of the S-RNase gene, were initially
used to amplify the sweet and sour cherry S-haplotypes simultaneously (Yamane et al.,
2001). This primer set did not reliably amplify S2 or S13, so S-allele specific primers
were used to amplify these S-haplotypes (S2: Sonneveld et al., 2001; S13: Sonneveld et al.,
2003). Pru-C2 and PCE-R also did not distinguish between S9 and Sb, so S-haplotype
specific primers were used for crosses that contain both of these S-haplotypes (S9:
Sonneveld et al., 2003). Sb-specific primers did not exist and were designed from
previously obtained cDNA sequences (Hauck et al., 2002). Table 4.4 shows the
sequences and annealing temperatures for all PCR primers used in this study. A similar
PCR temperature profile, other than the annealing temperature, was used for all PCR
reactions: an initial denaturing step (94°C, 2.5 min) followed by 35 cycles of 94°C (30

sec), X°C (30 sec), 72°C, (90 sec) and a final elongation step (72°C, 5 min).
S-genotype or S-phenotype determination for hypothesis testing

Six micrograms of DNA from 93 sour cherry selections chosen for S1 or SC

evaluation was digested with HindIII (Boehringer Mannheim Biochemicals,

83

Table 4.4: DNA sequences, annealing temperature, and references for S-RNase

genotyping PCR primers used in this study.

 

 

Primer Sequence Annealing Reference‘I
(5 '-3 ') Temp. (°C)

PruC2 CTA TGG CCA AGT AAT TAT TCA AAC C 56 T
PCE-R TGT TTG TTC CAT TCG CYT TCC C Y
PaSZ-F TAC TTC GAG CGA TCC CAA A 50

PaSZ-R AAG TGC AAT CGT TCA TTT G S
PaS9-F TT TGT TAC GTT ATG AGC AGC AG 62 R
PaS9-R ATG AAA CAA TAC ATA CCA CTT TGC TA R
PaSl3-F CA ATG GGT CGC AAT TTG ACG A 66 R
PaSl3-R GGA GGA GGT GGA TTC GAA CAC TTG R
Pch-F CAC CTG CAT ACT TCG CAA GA 66

Pch-R TGC TGC TTT AAT GGG TGC TA
‘ T (Tao et al., 1999); Y (Yamane et al., 2001); S (Sonneveld et al., 2001); R (Sonneveld
et al., 2003)

84

Indianapolis), resolved on a 0.9 % agarose gel for 36 h at 30 V, and transferred to a nylon
membrane (Hybond-N+, Amersham) according to Wang et al. (1998). PCR amplified
fragments of the S6-RNase cDNA from sweet cherry (Tao et al., 1999) were used as the
probe. This probe has been shown to cross-hybridize with all sweet and sour cherry S-
RNases (Tao et al., 1999; Yamane et al., 2001; Hauck et al., 2002). Probes were
radiolabelled with 32P-dCTP (DuPont, Boston) using the random primer hexamer-priming
method described by Feinberg and Vogelstein (1983). After hybridization at 60°C for 16
hours and high stringency washes (2 X 30 min with 2 X SSC and 1 % SDS followed by 2
X 30 min with 0.2 X SSC and 0.5 % SDS at 60 °C), radioactive signal was detected on X-

ray films using a Kodak RP X-OMAT processor.

Analysis of pollen tube growth for hypothesis testing
The 93 sour cherry selections were tested for S1 or SC by observing pollen tube

growth in self-pollinated styles. Pollination tests were performed as described by Lansari
and Iezzoni (1990) with slight modifications. Pollen fi'om newly opened flowers was
collected and dried for at least 24 h. Twenty flowers at the balloon stage were
emasculated. Ten emasculated flowers were hand pollinated with self-pollen when
receptive (24 h after emasculation) and ten flowers were pollinated with a mixture of
pollen from ten random sour cherry selections (“bulk pollen”). The pollinated pistils were
collected 72 h after pollination, placed in fixing solution [(1 chloroform: 3 (95%) ethanol:
1 glacial acetic acid) (v/v)] for 24 h, transferred into 100% ethanol, and stored at 4 °C
until used for observation. The pistils were washed thoroughly in tap water, incubated in

10 N NaOH for 4 h to soften the tissues, and soaked in 0.1 % aniline blue solution with

85

33 mM K3PO4 (pH 8.5) for 45 min to stain the pollen tubes. Pollen tubes were observed

by ultraviolet fluorescent microscopy (BX60, Olympus, Tokyo, Japan).

Results

Inter-specific crosses

Reciprocal inter-specific crosses between sweet and sour cherry were designed to
take advantage of their shared S-haplotypes to systematically test the functionality of the
sour cherry S4, S6 and Sty-haplotypes. The sweet cherry testers were Schmidt or Emperor
Francis (EF) (S4), Gold (S6) and Chelan (S9). The segregation of every S-haplotype in
each of the inter-specific populations is summarized in Table 4.5. The segregation data
that is most critical for dissecting the genetic control of S1 in sour cherry is described

below. Schematic representations of these crosses can be found in the Appendix (Figure

3.2).

Surefire x Emperor Francis (EF): All 30 progeny contained the S 3-haplotype; thus all
progeny resulted from fertilization of Surefire by EF pollen that contained the S 3-
haplotype. This indicates that pollen containing the S4 pollen-S products were recognized

and destroyed in a S-haplotype-specific manner by a functional S4-RNase in Surefire

styles.

86

 

 

 

 

 

 

 

 

 

 

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93

Emperor Francis (EF) x Surefire: Both the S 3- and S4-haplotypes segregated 1 present: 1
absent in the 37 progeny, and no progeny inherited both the S 3 and S4-haplotypes. These
findings indicate that Surefire pollen containing the fimctional S4 pollen-S product was
rejected in a S-haplotype-specific manner by the S4-RNase in the EF styles. The S13~
haplotype was present in nearly all progeny because it normally pairs with the S4-
haplotype during meiosis I. Since S4-containing pollen is specifically rejected, all
successful pollen will contain its homolog, S 1 3', unless tetrasomic inheritance occurred to

allow the creation of 5on pollen.

Ulfehérto'ifiirtos (UF) x Schmidt: All 45 progeny contained the S 2-haplotype, and
therefore all progeny resulted from fertilization of UF by Schmidt pollen that contained
the S 2-haplotype. This indicates that pollen containing the S4 pollen-S product were

recognized and destroyed in a S-haplotype-specific manner by a functional S4-RNase in

UF styles.

Emperor Francis (EF) x Ulfehérto'ifi‘irtos (UF): Both the S 3- and S4-haplotypes
segregated 1 present: 1 absent in the 36 progeny, and no progeny inherited both S 3 and
S4-haplotypes. These findings indicate that UF pollen containing the fimctional S4
pollen-S product was rejected in a S-haplotype—specific manner by the S4-RNase in the
EF styles. The Se-haplotype was present in nearly all progeny because it normally pairs
with the S4-haplotype during meiosis I. Since S4-containing pollen is specifically
rejected, all successful pollen will contain its homolog, Se, unless tetrasomic inheritance

occurred to allow the creation of 5de pollen.

94

Erdi Bo'termo" (EB) x Emperor Francis (EF): All ten progeny contained the S 3-haplotype,
thus all progeny resulted from fertilization of EB by EF pollen that contained the S 3-
haplotype. This indicates that pollen containing the S4 pollen-S product were recognized
and destroyed in a S-haplotype-specific manner by a fiJnctional S4-RNase in EB styles.
The cDNA sequence of the S4-RNase from EB was previously shown to be identical to
the S4-RNase of sweet cherry (Hauck et al., 2002), thus the finding that they are

functionally identical is not surprising.

Emperor Francis (EF) x Erdi Bo'termo" (EB): Both the S 3- and S4-haplotypes segregated 1
present: 1 absent in the 20 progeny, and no progeny inherited both the S 3 and S4-
haplotypes. These findings indicate that EB pollen containing the functional S4 pollen-S
product was rejected in a S-haplotype-specific manner by the S4-RNase in the EF styles.
The San-haplotype was present in nearly all progeny because it normally pairs with the
S4-haplotype during meiosis I. Since S4-containing pollen is specifically rejected, all
successfill pollen will contain its homolog, S6,", unless tetrasomic inheritance occurred to

allow the creation of San pollen.

Erdi Bo'termo" (EB) x Gold: The S 3-haplotype segregated 1 present: 1 absent in the 33
progeny. The S‘s-haplotype segregated 3 present: 1 absent. These findings indicate that
all Gold pollen, regardless of whether they contain S 3 or Sg, are capable of fertilizing EB.
Gold contains a functional pollen-S6 gene; therefore, the inability of EB to reject S6

pollen indicates that the S6m-RNase of EB is not firnctional. This is consistent with the

95

previous characterization of the San-haplotype (Yamane et al., 2003). The San-haplotype
contains an insertion of approximately 2600 bp in the promoter region of the S-RNase,

which prevented its expression.

Gold x Erdi Bo'termo" (EB): Both the S3- and S6-haplotypes segregated 1 present: 1 absent
in the 14 progeny, and no progeny inherited both the S 3 and Sd-haplotypes. These
findings indicate that EB pollen containing the functional S6", pollen-S product was
rejected in a S-haplotype-specific manner by the Sg—RNase in the Gold styles. This is
consistent with the previous characterization of the S6m-haplotype (Yamane et al., 2003).
Whereas the San-RNase was not expressed, the expression pattern and DNA sequence of
the San-SFB was identical to the S6-SFB. The S4-haplotype was present in nearly all
progeny because it normally pairs with the San-haplotype during meiosis I. Since S6",-
containing pollen is specifically rejected, all successful pollen will contain its homolog,

S4, unless tetrasomic inheritance occurred to allow the creation of $an pollen.

Cigany 59 x Gold: The Sg-haplotype segregated 1 present: 1 absent in the 36 progeny,
and the Sg-haplotype segregated 3 present: 1 absent. Both of these findings indicate that
all Gold pollen, regardless of whether they contain S 3 or S6, are capable of fertilizing
Cigany 59. Gold contains a functional pollen-S6 gene; therefore, the inability of Cigany

59 to reject 56 pollen indicates that the S6c-RNase of Cigany 59, similar to the S6m-RNase

of BB, is not firnctional.

96

Gold x Cigany 59: Both the S 3- and S‘s-haplotypes segregated 1 present: 1 absent in the 40
progeny, and no progeny inherited both the S 3 and Sg—haplotypes. These findings indicate
that Cigany 59 pollen containing the firnctional 56c pollen-S product was rejected in a S-
haplotype-specific manner by the S6-RNase in the Gold styles. The Sg-haplotype was
present in nearly all progeny because it normally pairs with the Sex-haplotype during
meiosis I. Since S6c-containing pollen is specifically rejected, all successful pollen will
contain its homolog, S9, unless tetrasomic inheritance occurred to allow the creation of

SaSb pollen.

Cigany 59 x Chelan: All seven progeny contained the S 3—haplotype, and therefore all
progeny resulted from fertilization of Cigany 59 by Chelan pollen that contained the S 3
pollen-S product. Thus, pollen containing the 59 pollen-S product were recognized and

destroyed in a S-haplotype-specific manner by a functional Sp-RNase in Cigany 59 styles.

Rheinische Schattenmorelle (RS) x Gold: All 31 progeny contained the S 3-haplotype, and
therefore all progeny resulted from fertilization of RS by Gold pollen that contained the
S 3-haplotype. Pollen containing the S6 pollen-S product were recognized and destroyed
in a S-haplotype-specific manner by a functional S6-RNase in RS styles. The cDNA
sequence of the S6-RNase from RS was previously shown to be identical to the S6-RNase

of sweet cherry (Hauck et al., 2002), thus the finding that they are fimctionally identical

is not surprising.

97

Gold x Rheinische Schattenmorelle (RS): Both the S 3- and S6-haplotypes segregated 1
present: 1 absent in the 13 progeny, and no progeny inherited both S 3 and Sg-haplotypes.
These findings indicate that RS pollen containing the functional S6 pollen-S product was
rejected in a S-haplotype—specific manner by the SyRNase in the Gold styles. The S13~
haplotype was present in all progeny because it normally pairs with the S6-haplotype
during meiosis I. Since S‘s-containing pollen is specifically rejected, all successful pollen

will contain its homolog, S13: unless tetrasomic inheritance occurred to allow the creation

of SaSb pollen.

Montmorency x Gold: All 55 progeny contained the S 3-haplotype, and therefore all
progeny resulted from fertilization of Montmorency by Gold pollen that contained the S 3-
haplotype. Thus, pollen containing the S6 pollen-S product were recognized and

destroyed in a S-haplotype-specific manner by a functional Sg-RNase in Montmorency

styles.

Gold x Montmorency: Both the S3- and S6-haplotypes segregated 1 present: 1 absent in
the 15 progeny, and no progeny inherited both S 3 and S6-haplotypes. These findings
indicate that Montmorency pollen containing the functional S6 pollen-S product was
specifically rejected in a S-haplotype-specific manner by the S6-RNase in the Gold styles.
The S 1 3~haplotype was present in nearly all progeny because it normally pairs with the
Sg-haplotype during meiosis I. Since Sg-containing pollen is specifically rejected, all
successful pollen will contain its homolog, S13; unless tetrasomic inheritance occurred to

allow the creation of $an pollen.

98

Sour cherry self-populations

The segregation of S-haplotypes in sour cherry self-populations was analyzed to
determine the functionality of S-haplotypes for which no sweet cherry tester was
available (S13, 50, Sb, S4 and Se). The segregation of every S-haplotype in each of the
self-populations is summarized in Table 4.6. The segregation data that is most critical for

dissecting the genetic control of SI in sour cherry is described below.

Rheinische Schattenmorelle (RS) self-population: Following self-pollination of RS, 54
progeny were genotyped to determine the segregation of the four S-haplotypes in RS.
The segregation of the S6- and Sb-haplotypes fit 1 present: 1 absent ratios, indicating the
presence of functional S-haplotypes in single copies. Pollen containing either of these
functional pollen-S products was likely rejected in a S-haplotype-specific manner by the
cognate functional S-RNases in RS styles. Both the Sa-and S13~haplotypes were present
in each of the 54 progeny, indicating that only pollen containing both So and 513' were
capable of self-fertilization. The S0 and 513' pollen-S products were not recognized and
degraded in a S-haplotype-specific manner by the S0 and S13' RNases, respectively. A

schematic representation of this self-pollination can be found in the Appendix (Figure

B.3).
Erdi Bo"termo" (EB) self-population: Following self-pollination of EB, eight progeny were

genotyped to determine the segregation of the three detectable S-haplotypes in BB. The

S4-haplotype segregated 1 present: 1 absent, indicating it is functional and present in a

99

Table 4.6: Sour cherry self-pollinations to test the filnctionality of S13 ., S0, S1,, S, S; and

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Se.
Rheinische Schattenmorelle (S6 S13’Sgfl self-pollinated
S-haplotype segregation Progeny phenotypes obtained
Expected Observed Chi Progeny No.
S-haplotym: ratio ‘ ratio square Probability Phenotype observed
'S6 1:1 28:26 0.02 0.89 S6 S13 -San 20
513- 1:1 54:0 52.0 <0.0001 S13-SaSbe 15
120 5420 - - S13'SanSx 11
Sa 1:1 54:0 52.0 <0.0001 S6S13'SaSb 8
1:0 54:0 - -
Sb 1:1 23:31 0.91 0.34
Erdi Botermo (S3993!) self-pollinated
S-haplotype segregation Progeny phenotypes obtained
Expected Observed Chi Progeny No.
S-haplotype ratio a ratio square Probability Phenotype observed
S4 121 424 0.13 0.72 S4S6mSan 4
S6," 1 10 810 - - S6mSanSx 4
So 1:1 8:0 6.13 0.01
1:0 8:0 - -
(guy 59 (SgtggSng) self-pollinated
S-haplotype segregation Progeny phenotypes obtained
Expected Observed Chi Progeny No.
S-haplotype ratio ‘ ratio square Probability Phenotype observed
Sac l :0 37:0 - - SaSaSbe 14
S9 1:1 ’ 14:23 1.73 0.19 S6oS9SaSb 9
Sa 1:1 37:0 35.0 <0.000l S6cSanSx 9
1:0 37:0 - - 56,599an 5
Sb 1:1 23:14 1.73 0.19
Surefire (S4 S1385!) self-pollinated
S-haplotype segregation Progeny phenotypes obtained
Expected Observed Chi Progeny No.
S-haplotype ratio ‘ ratio square Probability Phenotype observed
S4 1:1 21:15 0.69 0.41 S, S13'San 21
Sa 1:1 36:0 34.0 <0.0001 S13'SaSpr 15
1:0 36:0 - -
S13. 1:1 36:0 34.0 <0.0001
1:0 36:0 - -

100

(Continued)
Montmorency (S6 S13'S.S,") self-pollinated

 

 

 

 

 

S-haglotype segregation Progeny phenotypes obtained
Expected Observed Chi Progeny No.
S-haplotype ratio a ratio square Probability Phenotype observed
So 1 :1 72:63 0.47 0.49 56513 '5an 67
Sa 1:1 131:4 117.6 <0.0001 S13’SanSx 60
1:0 131:4 - - S6SI3'SxSx 3
513' 1:1 131:4 117.6 <0.0001 S6SaSpS'x 2
1 :0 13 l :4 - - SanSnS'x 2
513 '5»?an 1

Ilfl'ehértoi fiirtiis (S3995!) self-pollinated

 

 

 

 

 

S-ha lotype segregation Progeny phenotypes obtained
Expected. Observed Chi Progeny No.
S-haplotype ratio ' ratio square Probability Phenotype observed

S4 1:1 60:42 2.83 0.09 S¢SdSeSx 57
S4 1:1 98:4 84.8 <0.0001 SdSeSxSx 41

1:0 98:4 - - S4SeSxSx 3
Se 1: 1 102:0 100.0 <0.0001 SeSxSxSx 1

1:0 102:0 - -

 

“ Each S-haplotype was first tested for fit to a segregation ratio expected for a functional
S-haplotype present in a single copy (1 present: 1 absent). Ifrejected, the segregation
was tested for fit to a ratio expected for a non-filnctional S-haplotype (1 :0).

b For those sour cherry trees which have fewer than four different S-haplotypes, SC is used

to designate the fourth S-haplotype. Sx may either represent a S-haplotype in double

dose or a null allele.

101

single copy. Pollen containing the fiinctional pollen-S4 product was likely rejected in a S-
haplotype-specific manner by the firnctional S4-RNase in EB styles. Both the S6,,,- and
Sa-haplotypes were present in each of the eight progeny, indicating that only pollen
containing both S6", and S, were capable of self-fertilization. The S6", and Sa pollen-S
products were not recognized and degraded in a S-haplotype—specific manner by the S6",
and Sa RNases, respectively. A schematic representation of this self-pollination can be

found in the Appendix (Figure B.4).

Cigany 59 self-population: Following self-pollination of Cigany 59, 37 progeny were
genotyped to determine the segregation of the four detectable S-haplotypes in Cigany 59.
The segregation of the S9- and Sb-haplotypes fit 1 present: 1 absent ratios, indicating the
presence of filnctional S-haplotypes in single copies. Pollen containing either of these
functional pollen-S products was likely rejected in a S-haplotype—specific manner by the
cognate functional S-RNases in Cigany 59 styles. Both the Swand Sa-haplotypes were
present in each of the 37 progeny, indicating that only pollen containing both S6c and S,
are capable of self-fertilization. The Sgc and Sa pollen-S products were not recognized
and degraded in a S-haplotype-specific manner by the S60 and Sa RNases, respectively. A

schematic representation of this self-pollination can be found in the Appendix (Figure

13.5).

Surefire self-population: Following self-pollination of Surefire, 36 progeny were
genotyped to determine the segregation of the three detectable S-haplotypes in Surefire.

The S4-haplotype segregated 1 present: 1 absent, indicating it is functional and present in

102

a single copy. Pollen containing the functional pollen S4- product was likely rejected in a
S-haplotype-specific manner by the functional S4-RNases in Surefire styles. Both the Sa-
and S 1 3~haplotypes were present in each of the 36 progeny, indicating that only pollen
containing both 5,, and S13' are capable of self-fertilization. The S0 and S13' pollen-S
products were not recognized and degraded in a S-haplotype-specific manner by the S0
and S13o RNases, respectively. A schematic representation of this self-pollination can be

found in the Appendix (Figure B.6).

Montmorency self-population: Following self-pollination of Montmorency, 135 progeny
were genotyped to determine the segregation of the three detectable S-haplotypes in
Montmorency. The S6-haplotype segregated 1 present: 1 absent, indicating it is
functional and present in a single copy. Pollen containing the functional pollen Sa-
product was likely rejected in a S—haplotype-specific manner by the firnctional S6-RNases
in Montmorency styles. Both the Sa- and S 1 3~haplotypes segregated 131 present: 4
absent. The majority of progeny inherited both So and S13, indicating that the S0 and S13'
pollen-S products could not be recognized and degraded in an S-haplotype-specific
manner by the S0 and S13' RNases, respectively. However, four progeny were the result
of fertilization by pollen containing S0 and S,, and another four progeny were the result of
fertilization by pollen containing S13' and Sx, suggesting that the Sx-haplotype is also non-
functional, but possibly linked to a gene that is deleterious for pollen growth, thus making
pollen tubes containing 5,; less competitive. A schematic representation of this self-

pollination can be found in the Appendix (Figure B7).

103

Ulfehérto'iffirtos (UF) self-population: Following self—pollination of UF, 102 progeny
were genotyped to determine the segregation of the three detectable S-haplotypes in UF.
The S4-haplotype segregated 1 present: 1 absent, indicating it is fimctional and present in
a single copy. Pollen containing the fimctional pollen S4- product was likely rejected in a
S-haplotype-specific manner by the filnctional S4-RNases in UF styles. The Se-haplotype
was present in each of the 102 progeny whereas Sd segregated 98 present: 4 absent. The
majority of progeny inherited both S4 and Se, indicating that the 5,; and Se pollen-S
products could not be recognized and degraded in an S-haplotype-specific manner by the
5.; and Se RNases, respectively. However, four progeny were the result of fertilization by
pollen containing Se and Sx, suggesting that the Sx-haplotype is also non-functional, but
possibly linked to a gene that is deleterious for pollen growth, thus making pollen tubes
containing Sx less competitive. A schematic representation of this self-pollination can be

found in the Appendix (Figure B.8).

Hypothesis verification

These genetic analyses led to the formation of a hypothesis for the genetic control
of SI and SC in sour cherry, stating that a match between a fimctional pollen-S product in
the pollen and a firnctional S-RNase in the style will result in rejection of the pollen.
Pollen rejection will occur whether there are one or two fiinctional matches. However, if
there are no functional matches, the pollen will not be rejected. To test this hypothesis,
the SI or SC phenotypes of 92 sour cheny selections was determined via observation of

pollen tube growth down self-pollinated styles and compared with predictions based on

104

their S-genotypes. The results are summarized in Table 4.7. Ninety-one of the 92

predictions were accurate. The one inaccurate prediction has not been replicated.

Discussion

The segregation of S—haplotypes in various inter-specific crosses and self-
populations was analyzed to determine the fimctionality of the S-haplotypes in six SC
sour cherry selections in order to gain information on the cause of the partial breakdown
of 81 in this tetraploid species. The data suggest that functional and non-functional S-
haplotypes are present in each of the examined SC trees. A hypothesis explaining the
genetic control of SI and SC in sour cherry was developed and verified through crossing
experiments. The implications of these findings are discussed below.

Three of the S-haplotypes, S4, 86 and Sg, were fully functional and identical to S-
haplotypes found in sweet cherry, as initially hypothesized based on RFLP banding
patterns (Yamane et al., 2001). However, the definitive proof that they are identical is
that they can carry out S-haplotype-specific rejection in crosses with trees containing the
same S-haplotype. Observation of pollen tube grth in reciprocal inter-specific crosses
between Crisana (S 1S4Sd) and Rainier (S 1S4) suggested that the S 1 and S4-haplotypes were
fully functional and identical to the sweet cherry counterparts (Hauck et al., 2002). The
observation of S-haplotype segregation in inter-specific crosses and self-pollinations
presented in the current study confirmed that these three S-haplotypes were functional

and identical to the sweet cheny counterparts.

105

Table 4.7 : The S-genotype, SI or SC predictions based on the S—genotype, and the SI or
SC phenotype of 92 sour cherry selections used to test the validity of the

hypothesis for the genetic control of SI and SC in sour cherry.

 

 

Progeny Parents ‘ No. of SI/ SC SI/SC
S-genotype Individuals prediction phenotype

S13'SanSe UF x Sure 8 SC SC
S13vS,,S.,SJr UF x Sure 4 SC SC
S13'SanSx UF x Sure 2 SC SC
S¢S13'San UF x Sure 10 SC SC
5513.505. UF x Sure 3 so so
S4S13'San UF x Sure 2 SC SC
S13'SaSeSx UF x RS 1 SC SC
S13'SdSeSx UF x RS 4 SC SC
565136an UF x RS 2 SC SC

S13'SbSdSe UF x RS 1 SC SC
54563be UF x RS 1 SI SI
S4S6SbSd UF x RS 3 SI SI
SanSan UF x Mont 1 SC SC
S13'SarSdSJIC UF x Mont 1 SC SC
SeSdSeSx UF x Mont 3 SC 2 SC, 1 SI
S4SgSaSe UF x Mont 1 SC SC
$66813de UF x Mont 1 SC SC
5455an UP x Mont 2 SI SI
545135051, RS x BB 4 SC SC
$45135an RS x EB 17 SC SC
S4S6Sl3’Sa RS x BB 8 SC SC
S4S6SaSb RS x EB 5 SI SI
54565be RS x BB 8 SI SI

 

‘ UF = Ujfehértoi fiirtos; Sure = Surefire; RS = Rheinische Schattenmorelle; Mont =

Montmorency; EB = Erdi B6term6

106

The segregation of the Sb-haplotype in the RS and Cigany 59 self-populations
indicate that it is also a fully functional allele that can carry out S—haplotype specific
rejection. Although, to date, we have not found an identical S-haplotype in sweet cherry,
it is possible that the Sb-haplotype does exist in wild sweet cherry gerrnplasm.
Alternately, it is possible that this S-haplotype was inherited from the other parent of the
allotetraploid sour cherry, P. fruticosa. There has not been an in depth investigation of SI
in P. fiuticosa, making it currently impossible to know whether the Sb-haplotype is
originally from sweet cherry or P. fruticosa. A third possibility is that this S-haplotype
was formed more recently in sour cherry, and does not exist in either of the progenitor
species.

Each of the six SC selections included in this study contain at least one functional
S-haplotype that is capable of carrying out S-haplotype—specific rejection of pollen. RS
contains both S6 and Sb, Cigany 59 contains Sg and Sb, EB contains S4, Surefire contains
S4, UF contains S4 and Montmorency contains S6. This implies that each of these
selections contains all the necessary machinery for initiating and carrying out an SI
reaction. Thus, the breakdown of SI in these plants was not due to a mutation of one of
these components, but instead must have been caused by a mutation in one of the S-
haplotype specificity components, either the S-RNase or the pollen-S.

In addition, RS (S6 and S), Cigany 59 (S9 and Sb), and EB (S4 and S6,") contain
two S-haplotypes with firnctional pollen-S genes. Thus, it would be expected that each of
these three SC selections would produce some pollen that contains two fimctional pollen-
S genes. If the breakdown of SI in sour cherry were caused by the competition of pollen-

S products in heteroallelic pollen, then these pollen types would be able to successfully

107

fertilize any styles, including those containing the counterpart S-RNases. However, in
each case, these S-haplotypes were never inherited by progeny via pollen in these crosses.
For instance, when RS was used to pollinate Gold styles, none of the progeny inherited
the S6 haplotype from RS. This result indicates that pollen containing S6 was specifically
rejected in Gold styles, regardless of whether there was only one functional pollen-S
product (either $6.90 or S; 513') or two functional pollen-S products (S6Sb). Two
hypotheses that could defend the competitive interaction theory are that either gametes
containing S6 and Sb never form due to the pairing of chromosomes during meiosis or
these gametes are not viable due to the linkage of one of these S-haplotypes to a
deleterious gene. However, in the reciprocal cross in which Gold was used to pollinate
RS, 8 out of 31 progeny inherited the S6 and Sb from RS. Approximately one quarter of
the progeny inherited this S-haplotype combination from RS, which would be expected
assuming strict disomic inheritance of alleles that are on homoeologous chromosomes.
Thus, gametes containing this combination of S-haplotypes occur at a high frequency.
The fact that no RS pollen containing the S6-haplotype was inherited in the Gold x RS
cross suggests that heteroallelic pollen retains its ability to trigger an SI reaction and that
SI does not breakdown in sour cherry due to a competition between pollen-S products in
heteroallelic pollen.

Since the breakdown of SI in sour cheny is not caused by competition between
pollen-S products within a pollen tube or by mutations in non-haplotype-specific modifier
genes, an alternate explanation for the observed breakdown is necessary. The data
suggests that the breakdown of SI in sour cherry is due to the accumulation of non-

functional S-haplotypes that have lost the fiinction of at least one of the S-haplotype

108

specificity components, S-RNase or pollen-S. Several S-haplotypes, specifically S60, S6,”,
S13, Sa, S1, and Se, all appear to be non-functional. None of these S-haplotypes is capable
of initiating an SI reaction, even if a corresponding S-haplotype was also present in the
stylar parent.

In the case of S6,» and S6,", it was possible to use reciprocal crosses with Gold,
which is known to contain a fully functional Syhaplotype, to determine if the S-RNase
and/or the pollen-S gene were non-fiinctional. As summarized in Table 4.5, when Gold
was the maternal parent and either Cigany 59 or EB was the paternal parent, none of the
progeny inherited a gamete containing either S6c or S6", from Cigany 59 or EB,
respectively. This result suggests that the Sg—RNase in the Gold styles was able to
specifically reject pollen containing either the S6c- or San-haplotype. Thus the pollen-S
product must be functional and capable of triggering the SI reaction in both of these
van'ants of the S6-haplotype. When Cigany 59 or EB was pollinated with Gold pollen,
the S 3-haplotype segregated 1:1 whereas the SSC and S6m-haplotypes each segregated 3: 1.
This result suggests that pollen containing either S 3 or S; was able to grow through
Cigany 59 or EB styles. Gold contains a functional pollen-S6 gene, so the ability of S6
pollen to successfirlly pollinate Cigany 59 or EB must be due to the S6c- and San-RNase
genes being non-functional. Yamane et a1. (2003) characterized the S6m-haplotype from
EB and discovered that the S6m-RNase is not expressed in BB styles, probably because of
a 2600 bp insertion in the putative promoter region of the ng-RNase gene. The sequence
and expression of the ng-SFB was identical to that of the S6-SFB from sweet cherry.

Similar sequence and expression analyses of the S6c-haplotype from Cigany 59 are

underway.

109

It should also be possible to determine if the S-RNase or pollen-S gene is non-
functional in the S13~haplotype, since a functional counterpart exists in sweet cherry;
however, appropriate crosses were not made due to the lack of availability of a S13 sweet
cherry tester. Tobutt et al. (2004) reported that the sour cherry selections Marasca
Piemonte, Marasca Savena and Morello Dutch, which all contain S6 and S13, reject pollen
from the sweet cherry Noble (56.313). This suggests that the 513 from these sour cherry
selections contain a functional S13-RNase that is similar to the S13 from sweet cherry. The
reciprocal cross, however, was not reported. These results make it likely that S13' contains
a functional S13-RNase but a non-firnctional pollen-S13 gene. Molecular analysis of the
S 1 3~haplotype is underway.

Since functional versions of the S0, S; and Se-haplotypes are not known in sweet
cherry, self-pollinations were used to conclude that each of these S-haplotypes were non-
functional. An example of how this data is interpreted is as follows. The S-genotype of
RS is S6 S13'SaSb. In the self-progeny, both the S6 and Sb haplotypes segregate 1: 1,
whereas every progeny inherited both the So and S13'haplotypes. RS styles are
hypothesized to contain firnctional S6- and Sb-RNases; however, the presence of
functional Sa- or S 1 3~RNases has not been confirmed. Since sour cheny is a segmental
allopolyploid capable of occasional multivalent formation (Wang et al., 1998), six pollen
types are possible from RS: S650, S6Sb, S6 513', SaSb, S0 513', and Sb S132 Both the S6 and Sb
pollen-S products are functional; however, the presence of a functional pollen-S product
for So and S13' is not known. It is hypothesized that either the S-RNase or pollen-S gene
for each of these two non-firnctional S-haplotypes is mutated, preventing specific

rejection of these S-haplotypes. Since the S6- and Sb-haplotypes are functional, any

110

pollen containing one or both of these S-haplotypes will be recognized and destroyed by
the S6- or Sb-RNases in the styles, respectively. Any pollen that does not contain either S6
or Sb will not be recognized. Thus, all self-progeny should, and did, inherit the Sa- and
S 1 3~haplotypes fiom pollen. Similar analyses were done for the S4- and Se-haplotypes.

Whereas the self-pollinations allowed the conclusion that S0, S4 and S. are all non-
functional S-haplotypes, it would be necessary to find a functional counterpart to these S-
haplotypes, either in sweet or sour cherry, before conclusions can be drawn as to whether
mutations in the S-RNase or pollen-S gene cause a loss-of-function. Since functional
versions of the S,,, S4 and Se-haplotypes are currently undiscovered in sweet cherry, inter-
specific crosses could not be used to determine whether the S-RNase and/or the pollen-S
gene was non-functional. However, the cDNA and N-terminal amino acid sequences
were reported for the Sa-RNase (Yamane et al., 2001; Hauck et al., 2002), suggesting that
this protein is made. Although the filnctionality of the Sa-RNase is unknown, the fact that
it is expressed suggests-that the loss-of-function of the Sa-haplotype may be due to
mutations in the pollen-S gene. We are currently in the process of cloning and
sequencing the S-RNase and SFB genes, as well as their promoters, for the 50, S4 and Se-
haplotypes to look for mutations. In addition, expression analyses will be conducted for
both genes in each of these S-haplotypes. These types of molecular characterizations,
similar to those done for the San-haplotype (Yamane et al., 2003), should tell us which of
the genes has been mutated in each of these non-functional S-haplotypes.

In this study, two selections, Cigany 59 and EB, contain non-functional S6-
haplotypes composed of a functional pollen-S gene and a non-functional S—RNase gene.

Existing evidence suggests that these mutations were caused by independent events.

111

First, RFLP analysis following digestion with HindIII and probing with an S-RNase
probe revealed different banding patterns for Sgc and 56m (Yamane et al, 2001). The
fragment corresponding to S6,- was similar in size to that of the fiinctional S6-haplotype
from sweet cherry (approximately 5.8 kb), whereas the S6", fragment was approximately 9
kb. Thus, it is likely that the loss-of-function is not caused by a large insertion, but rather
a smaller insertion, deletion or a base substitution. In addition, PCR primers designed to
amplify the 2600 bp insertion in the San-RNase promoter (Yamane et al., 2003) do not
amplify a band in Cigany 59 (data not shown). The occurrence of two independent
mutations in the Sg—RNase gene in sour cherry could imply the presence of a mutational
“hot spot”. It is interesting to note that the distance between the Sg-RNase and Sg-SFB is
only approximately 300 bp (Yamane et al., 2003), compared to other S-haplotypes that
may have intergenic spaces as great as 40 kb (K. Ikeda, unpublished).

In four of the six sour cherry studies examined, only three S-haplotypes could be
distinguished. In addition, the segregation data suggested that each of these S-haplotypes
was present in a single copy. Thus, the fourth S-haplotype in these selections is
hypothesized to be a null allele (Snuu), consisting of a large deletion that included the S-
RNase. The deletion of the S-RNase is consistent with the presence of only three
fragments on a Southern upon hybridization with an S-RNase probe. Sm,” is expected to
be non-functional due, at least, to the absence of a fiinctional S-RNase. However, it is
unknown whether the deletion includes the pollen-S gene. The presence of a functional

pollen-S gene would allow pollen containing the Snuu to be rejected by styles containing a

cognate functional S—RNase, if one exists.

112

The ability of heteroallelic sour cherry pollen to trigger an SI reaction suggests
that the identity of the pollen-S gene and/or the mechanism of SI in the Solanaceae may
differ from that in Prunus, despite the belief that the S-RNase in the Solanaceae and
Rosaceae are orthologous (Igic and Kohn, 2001) and the recent discovery that an F-box
gene may be the pollen determinant in both the Solanaceae and Prunus (Entani et al.,
2003; Ushijima et al., 2003; Sijacic et al., 2004). However, initial comparison of the
different F-box genes suggests that they are not orthologous. In all species studied to
date, multiple F -box genes are linked to the S-RNase; thus, it is possible that Prunus and
the Solanaceae recruited different F-box genes (SFB vs PiSLF) to act as the pollen-S
gene. An additional difference between gametophytic S1 in Prunus and the Solanaceae is
that, despite the identification of several PPMs through mutant screens, no PPMs caused
by either the loss of expression of the pollen-S gene or the expression of a mutant pollen-
S gene have been observed in the Solanaceae (Pandey 1967 ; van Gastel and de
Nettancourt, 1975; Golz et al., 1999; Golz et al., 2000). Instead, all identified
Solanaceous PPMs were caused by the duplication of the pollen-S gene. In comparison,
at least two PPMs in Prunus, S4v from sweet cherry and Sf from P. mume (U shijima et al.,
2004) have been caused by mutation of the pollen-S gene. These findings suggest that
the identity and function of the pollen-S gene may differ between the Solanaceae and
Prunus. F urtherrnore, competitive interaction between pollen-S products is thought to
cause a breakdown of SI in other Rosaceous genera, such as Malus and Pyrus (R. Tao,
pers. comm). Therefore, it is possible that not only does the identity and function of the

pollen-S gene differ between the Solanaceae and Prunus, but also between Prunus and

other Rosaceous genera.

113

The data presented in this research indicate that the cause of the partial
breakdown of SI in sour cherry is due to the accumulation of non-functional S-haplotypes
that contain mutations in one or both of the S-haplotype-specificity components, S-RNase
or pollen-S, rather than competitive interaction between pollen S-products or due to
mutations in a modifier gene. Therefore, our current hypothesis for the genetic control of
SI and SC in sour cherry is that a match between a functional pollen-S product in the
pollen and a functional S-RNase in the style will result in rejection of the pollen. Pollen
rejection will occur whether there are one or two functional matches. However, if there
are no functional matches, the pollen will not be rejected.

To test the validity of this hypothesis, the SI or SC phenotypes of 92 sour cherry
selections was determined via observation of pollen tube grth in self-pollinated styles
and compared with predictions based on their S-genotypes. Of these 92 predictions, 91
were accurate. The one incorrect prediction was for a selection that had the S-genotype
S 1 3'SdSan. The presence of at least two non-firnctional S—haplotypes in this selection
would make this tree SC, according to the hypothesis. The reason for the incorrect
prediction is unclear and is currently being investigated. However, this result has not
been replicated and the most likely explanation is that the genotyping and phenotyping
were mistakenly conducted for different trees. This experiment will be replicated in the
Spring of 2005.

Previously, the segregation of S-haplotypes and the SI or SC phenotype among
progeny in a cross between RS and EB was reported (Hauck et al., 2002). At the time,
we could not explain the observed segregation. However, with our current understanding

of the firnctionality of the S-haplotypes in these two parents and our hypothesis

114

explaining the genetic control of SI and SC in sour cherry, we can now explain the
observed segregation. All progeny that contained at least two non-fimctional S-
haplotypes were SC, whereas all progeny that contained fewer than two non-functional S-
haplotypes were SI. See Table B3 and Figure B] for a complete list of progeny S-
genotypes and SI or SC phenotypes.

The elucidation of the genetic control of SI and its breakdown in sour cherry has
implications on our understanding of gametophytic SI and the effects of polyploidy on
gametophytic SI, as discussed above, as well as on sour cherry breeding. Growers
demand SC sour cherry cultivars due to inefficiencies related to growing SI cultivars,
such as a reliance on bees for fiuit set and the need to grow pollinator varieties in the
orchard. Thus, one trait that breeders are concerned with is SC. With the knowledge of
the genetic control of SI and SC in sour cherry, a breeder could use S-haplotype markers
to predict whether a seedling is S1 or SC, allowing the elimination of seedlings that are
predicted to be SI. In addition, the breeder could predict whether a particular cross would

result in SI progeny, SC progeny or a mixture of both types of progeny. Thus, breeders

could save both time and orchard space.

115

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glycoprotein associated with expression of self-incompatibility in Nicotiana alata.
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Pandey KK (1968) Colchicine induced changes in the self-incompatibility behaviour of
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(2004) Identification of the pollen determinant of S-RNase-mediated self-
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incompatibility alleles and development of allele-specific PCR detection. Theor Appl
Genet 102:1046-1055.

Sonneveld T, Tobutt KR, Robbins TP (2003) Allele specific PCR detection of sweet

cherry SI (S) alleles S1 to S16 using consensus and allele specific primers. Theor Appl
Genet 107: 1059-1070.

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118

Tobutt KR, Boskovic R, Cerovic K Sonneveld T, Ruzic D (2004) Identification of
incompatibility alleles in the tetraploid species sour cherry. Teor Appl Genet
1082775-785.

Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H (2003) Structural
and transcriptional analysis of the self-incompatibility locus of almond: identification

of a pollen expressed F -box gene with haplotype-specific polymorphism. Plant Cell
1 5:77 1 -7 8 1 .

Ushijima K, Yamane H, Watari A, Kakehi E, Ikeda K, Hauck NR, Iezzoni AF, Tao R
(2004) The S haplotype-specific F-box protein gene, SFB, is defective in self-
compatible haplotypes of Prunus avium and P. mume. Plant J (in press).

van Gastel AJ G, de Nettancourt D (1975) The effects of different mutagens on self-
incompatibility in Nicotiana alata Link and Otto. H. Acute irradiation with X-rays
and fast neurons. Heredity 34:381-3 92.

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using RFLP markers. Theor Appl Genet 97:1217-1224.

Wiersma PA, Wu Z, Zhou L, Hampson C, Kappel F (2001) Identification of new self-
incompatibility alleles in sweet cherry (Prunus avium L.) and clarification of

incompatibility groups by PCR and sequencing analysis. Theor Appl Genet 102:700-
708.

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of four self-incompatibility alleles in sweet cherry (Prunus avium L.). Theor Appl
Genet 108:299-305.

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characterization of S-RNases in tetraploid sour cherry (Prunus cerasus L.). J Amer
Soc Hort Sci 126:661-667.

Yamane H, Ikeda K, Hauck NR, Iezzoni AF, Tao R (2003) Self-incompatibility (S) locus
region of the mutated S6-haplotype of sour cherry (Prunus cerasus) contains a

functional pollen S allele and a non-functional pistil 8 allele. J Exper Bot 54:243]-
243 7.

119

CHAPTER 5

CONCLUSIONS, GENERAL DISCUSSION
AND FUTURE RESEARCH

120

Conclusions and General Discussion

The goal of the presented research was to determine the genetic control of self-
incompatibility (SI) and self-compatibility (SC) in sour cherry. To accomplish this goal,
it was first necessary to become familiar with the S-haplotypes that exist in sweet cherry,
one of the progenitor species of sour cherry. RF LP analyses were used to determine the
S-haplotype constitution of 22 sweet cherry selections. In total, the RFLP banding
patterns were described for 14 different S-RNase alleles. Several new sweet cherry S-
haplotypes have since been reported [S 14: Wiersma et al., (2001); S16: Sonneveld et al.,
(2003); S23, S24, S25: Wunsch and Horrnaza (2004)]. As new S-haplotypes are reported, it
will be possible to compare them with the sour cherry S-haplotypes to see if they are
identical.

RFLP analyses were then conducted for a diverse set of 12 sour cheny selections
to determine which S-haplotypes exist in sour cherry (Yamane et al., 2001). A total of 11
different S-haplotypes were identified using two different restriction enzymes. Of these,
S1, S4, S6, S9 (called 5,.) and S 1 2 (called 813) had identical banding patterns to S-haplotypes
in sweet cherry, whereas S6,”, Sa, Sb, 5,, S; and Se had unique banding patterns. Whether
the S-haplotypes that appeared to be in common were functionally similar remained
unknown until reciprocal inter-specific crosses with sweet cherry were analyzed in this
study (discussed later). Since the release of this paper, the Sc has been renamed S13! due
to a report that several sour cherry trees contained the S13-RNase protein (Tobutt et al.,
2003) and the S-RNase amino acid sequences for Sc and 513 were identical. However, the

513' haplotype appears to be a non-fianctional version of S13, probably due to a mutation in

121

the pollen-S gene. In addition, the Se-haplotype is most likely a mutated version of S1,
containing a functional S 1-RNase and a non-firnctional pollen-S; gene. This conclusion is
based on the segregation of the S 1, S3 and Se, -haplotypes in reciprocal crosses with the
sweet cherry Regina (S 1S 3) and the ability to amplify a fragment from S, that is identical
to S1 using S j-RNase allele specific primers and S-RNase consensus primers.

Observations of pollen tube growth in inter-specific crosses involving the sour
cherry Crisana (S 1S4Sde) and the sweet cheny Rainier (S 1S4) provided the first evidence
that the S1 and S4-haplotypes in sour cherry, as identified by RFLP, were fully fiinctional
and identical to the S1 and S4 haplotypes in sweet cherry. The incompatibility of Crisana
pollen with Rainier styles also provided the first evidence that the breakdown of SI in
heteroallelic pollen observed in Solanaceous species does not occur in sour cherry, since
Crisana should be able to produce some pollen containing two functional pollen-S
products but is SI. If the competition between pollen-S products in heteroallelic pollen
causes a breakdown of SI in Prunus, then Crisana should be SC.

Finally, the segregation of S-haplotypes in various inter-specific crosses and self-
populations established that sour cherry is composed of a mixture of firnctional (S 1, S4, S6,
S9, and S) and non-firnctional (S66, S6,", S13; Sa, 54, and Se) S-haplotypes. In addition, all
sour cherry selections tested were capable of carrying out S-haplotype-specific rejection,
indicating that the breakdown of SI in sour cherry was not caused by mutations in the
machinery necessary to carryout an SI reaction, but rather in the specificity components,
either the S-RNase or the pollen-S gene, themselves. The possibility that competitive
interaction between pollen S-products causes the breakdown of SI in sour cheny was

eliminated because of the overwhelming evidence against this hypothesis in the

122

segregation data. Instead, the genetic cause of SC in sour cherry is the accumulation of
non-functional S-haplotypes that are incapable of triggering or initiating an SI reaction.

Taken together, the data support the “one-allele match” hypothesis. This
hypothesis states that the genetic control of SI and SC in sour cherry is based solely on
which S-haplotypes are present. Any time there is a match between a functional pollen-S
product in the pollen and a functional S-RNase in the style, the pollen will be rejected.
Pollen rejection will occur whether the pollen is homoallelic or heteroallelic and whether
there are one or two functional matches. However, if there are no functional matches, the
pollen will not be rejected.

These findings not only further our understanding of the genetic control of S1 in
sour cherry, they should aid sour cheny breeders and increase our knowledge of how

polyploidy affects GSI. These impacts are discussed in more detail below.

Impact on sour cherry breeding

In order for growers to obtain fruit from an SI variety, pollinator trees must be
inter-planted with the variety of interest and bees must be released to ensure proper cross-
pollination. These problems associated with growing SI trees make SC cultivars highly
desirable. Thus, breeders must select for SC trees in addition to improved fruit quality,
tree quality and resistance traits. Currently, breeders must wait three to five years before
flowers are available to test for SC. If the selection process could be done earlier, the
breeder could eliminate all SI material, saving space in their orchard and evaluation time
for seedlings that are SC. Until now, the genetic cause of SC was unknown, making it

impossible to use molecular markers to predict the SI or SC phenotype of a seedling.

123

Now that the genetic cause of SC is known, breeders can look for trees that contain at
least two of the identified non-functional S-haplotypes. Any seedling that contains fewer
than two non-functional S-haplotypes will be SI. With this information, breeders can
select for SC material immediately after germination of the seedling. In addition, a
breeder can select parents that maximize the chances of obtaining SC progeny. For
example, the pollination of a tree of S-genotype S4SaSng (which is SI) with pollen from a
tree that is S4SaSaSc would result in 100% SC seedlings, since the only successful pollen
would be SaSc, thus all progeny would also contain these two non-functional S-
haplotypes. Alternately, a cross between two SC trees, S1S4SoSd and SgSaSaSe, would
result in a mixture of SI and SC progeny. Without understanding the genetic basis of SI
and SC, the breeder might assume that the cross between two SC parents would result in
more SC progeny than the cross between the SI and SC parent.

Chapter 4 presented the results from SI or SC phenotypic predictions for 92 trees
from the MSU Clarksville Horticultural Research Station (CHES) based on their S-
genotypes or S-phenotypes. In total, accurate predictions were made for 91 of the 92
trees. The one incorrect prediction was for a progeny from a cross between Ujfehértoi
fiirtos (UF) and Montmorency that had the S-genotype 5.3.5.5... This tree contains at
least three non-firnctional S-haplotypes and should be SC, according to the “one-allele
match” hypothesis. The cause of the inaccurate prediction is currently being
investigated; however, it is very likely that this discrepancy was caused by lab error. For
example, it is possible that the S-genotyping and SI/SC phenotyping were mistakenly
conducted on different plants. However, a success rate of 91 out of 92 verifies the

effectiveness of screening seedlings for SC types. In addition, the compatibility of 13

124

crosses between trees located at CI-IES (Table B.2) were predicted. In this case, 100% of
the predictions were accurate.

One potential problem is if “new” germplasm is used for crosses. All the research
presented in this dissertation is based on the investigation of the S-haplotypes that are
present in 12 sour cherry selections. These 12 selections obviously do not contain every
S-haplotype that exists in sour cherry. Thus, the use of other sour cheny germplasm
could introduce new S-haplotypes. The firnctionality of these S-haplotypes is unknown,
making the prediction of S1 or SC difficult. Table B.1 includes nine progeny of trees that
have never been studied [111 18(12) and Erdi Jubileum]. Six progeny were from a cross
between UP and HI 18(12), which was found to have a new S-haplotype, named Sf.

Three addition progeny were from a cross between Montmorency and Erdi Jubileum,
which also contained a new S-haplotype, named Sg. For two progeny from these crosses
[27 9(12) and 27 8(5 8)], correct phenotype predictions could only be made if the Sf and S3
were assumed to be non-functional. The other seven progeny fiom these crosses could be
predicted without any assumptions since they contained more than two non-functional S-
haplotypes, regardless of whether or not the Sf and S8 are firnctional. In conclusion,
breeders can accurately use markers to predict the SI or SC phenotype of seedlings as
long as there is knowledge of the S-haplotypes that exist in the seedling. Novel S-

haplotypes could be problematic for accurate SI or SC phenotype prediction of seedlings.
Implications for understanding the efl'ect of polyploidy on GSI

Since the first observations of SC tetraploid plants in the 1930’s (Crane and

Lawrence, 1931), researchers have thought that GSI breaks down in response to an

125

increase in ploidy level. It was later hypothesized that the presence of two pollen-S
products in a single pollen tube results in a competition and a loss of SI phenotype
(Lewis, 1943). Recent experiments with Petunia verified that heteroallelic pollen loses
its SI phenotype (Entani et al., 1999). In fact, Sijacic et al. (2004) took advantage of the
competitive interaction phenomenon to prove that a recently cloned gene, PiSLF, was the
long sought-after pollen-S gene in Petunia inflata.

Stebbins (1950) predicted that SC is more common in polyploids because SC is
necessary for the establishment of an otherwise reproductively isolated plant (Stebbins,
1950; Miller and Veneble, 2000) and because polyploids can tolerate selfing due to less
inbreeding depression (Lande and Schemske, 1985). Husband and Schemske (1997)
provided evidence that the frequency of SC is higher in tetraploids than in diploids.
However, a more recent comparison of ploidy level and the SI or SC phenotype of
species among angiosperrns led to the conclusion that the frequencies are not
significantly different (Mable, 2004). These findings suggest that self-fertility is not a
prerequisite for the establishment of a polyploid, as previously suggested by Stebbins
(1950)

The results from the current study suggest that polyploidy itself does not directly
cause SC in sour cherry, but instead can have an indirect effect on SI. The presence of
six natural non-functional S-haplotypes in a set of 12 sour cherry selections is
surprisingly high compared to the diploid sweet cherry, which carries no known natural
non-flinctional S-haplotype. This suggests that the S-locus may be less stable in
polyploids than in diploids. Whether the loss-of-function occurred immediately after

polyploid formation due to an increase in transposition and other genome rearrangements

126

often seen in newly formed polyploids (Song et al., 1995; Shaked et al., 2001; Ozkan et
al., 2001) or if it is by on-going loss-of-function mutations is unclear; however, the
evidence suggests that the S-locus is less stable. Although the loss of GSI appears to be
directly caused by the increase in ploidy in the Solanaceae, the loss of GSI may not
always be directly caused by polyploidy, as evidenced by the current study of sour
cherry.

In the Solanaceae, SI breaks down in response to the competitive interaction
between pollen-S products in heteroallelic pollen. This phenomenon does not occur in
Prunus, suggesting that the identity of the pollen-S gene and/or the GSI mechanism may
differ between the Solanaceae and Prunus. This observed difference between the
Solanaceae and Prunus was unexpected, given that phylogenetic analyses of the S-RNase
gene suggest a common origin of the gene in the Solanaceae and Prunus, with the only
major difference being the S-RNase gene from Prunus contains two introns while the S-
RNase gene from all other Rosaceous species and all Solanaceous species contain only
one (Igic and Kohn, 2001).

Similar phylogenetic analyses for the pollen-S gene have not been conducted.
Initial comparison of the putative pollen-S gene from Prunus (SFB: Ushijima et al., 2003)
and from Antirrhinum (SFL: Lai et al., 2002) suggests that they are not orthologous.
Interestingly, no pollen-part mutant (PPM) caused by the mutation of the pollen-S gene
has been identified in the Solanaceae (Pandey 1967; van Gastel and de Nettancourt, 197 5;
Golz et al., 1999; Golz et al., 2000). Instead, all identified PPMs in the Solanaceae were
caused by the duplication of the pollen-S gene. In comparison, at least two PPMs in

Prunus, Sr from sweet cherry and Sf in P. mume (U shijima et al., 2004) have been caused

127

’71

by mutation of the pollen-S gene. These findings suggest that the identity and function of
the pollen-S gene may differ between the Solanaceae and Prunus. Thus, it is possible
that the Solanaceae and Prunus use a homologous style-deterrninant (S-RNase) but
recruited different genes to act as the pollen-detenninant (SFB vs SLF) in GSI.

Alternately, the breakdown of GSI due to competitive interaction between pollen-
S products might be more likely in autotetraploids than in allotetraploids. The studies in
the Solanaceae have mainly focused on artificially induced polyploids caused by the
addition of colchicine, whereas sour cherry is a natural allotetraploid species. It is
possible that the physical nature of autopolyploids and allopolyploids is the cause of the
observed difference in heteroallelic pollen. For example, it is possible that pollen-S gene
products from the different genomes of an allotetraploid do not interact and are not
capable of competition, whereas the pollen-S gene products from within the same

genome of an autotetraploids are capable of interacting and competing with one another.

Future Research

The presented research successfully determined the genetic control of SI and SC in sour
cherry. However, several other questions have emerged during the past few years.
Presented below is a list of questions that should be addressed and experiments that

should be conducted to help answer those questions.

128

1. What is the molecular basis for the loss-of-fiinction of each of the non-functional

sour cherry S-haplotypes (S60, S13, S0, S4 and Se)?

The following experiments will allow the detection of mutations in either the coding
region or regulatory region of the genes from each non-fimctional S-haplotype. This
is similar to the work done to characterize the loss-of-function mutation observed in

the San-haplotype from sour cherry (Y amane et al., 2003) and S4~haplotype and S,-

haplotypes fi'om sweet cherry and Japanese apricot, respectively (U shijima et al.,

2004)

1a: Determine the cDNA sequence of each S-RNase

1.b: Isolate genomic clones containing SFB and determine the DNA sequence of

SFB.

l.c: Use RT-PCR to determine whether each S-RNase or SFB is expressed in styles
or pollen, respectively.

1.d: For those genes that are not expressed, sequence the promoter region to

determine the cause of the observed loss of expression.

2. Are P. fiuticosa individuals SI or SC and what S-haplotypes exist in P. fiuticosa?

The existence of GSI in sweet cherry and sour cherry is well documented; however,

the existence of GSI in P. fruticosa, the other progenitor of sour cherry, has not been

documented. In addition, several sour cherry S-haplotypes have not been found in

129

sweet cherry and it is possible that they originate from P. fiuticosa. A complete
understanding of SI and its partial breakdown to SC in sour cheny depends on an
understanding of SI in P. fruticosa. The following set of experiments will give initial

insights on SI in P. fruticosa.

2A: Determine the SI or SC phenotype of a set of P. fiuticosa selections from a
diverse range of geographic locations.

2B: Use RFLP analyses to determine what S-haplotypes exist in this diverse set of
P. fiuticosa germplasm.

2C: Obtain cDNA sequences for S-RNases from P. fruticosa selections. Compare

the S-RNase sequences with those from sour cherry.

130

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incompatibility in Nicotiana alata Link and Otto. II. Acute irradiation with X-rays
and fast neurons. Heredity 34:381-3 92.

Wiersma PA, Wu Z, Zhou L, Hampson C, Kappel F (2001) Identification of new self-
incompatibility alleles in sweet cherry (Prunus avium L.) and clarification of

incompatibility groups by PCR and sequencing analysis. Theor Appl Genet 102:700-
708.

Wunsch A, Hormaza II (2004) Cloning and characterization of genomic DNA sequences

of four self-incompatibility alleles in sweet cherry (Prunus avium L.). Theor Appl
Genet 1082299-305.

Yamane H, Tao R, Sugiura A, Hauck NR, Iezzoni AF (2001) Identification and

characterization of S-RNases in tetraploid sour cherry (Prunus cerasus L.). J Am Soc
Hort Sci 126:661-667.

132

Appendices

133

APPENDIX A

DNA EXTRACTION PROTOCOL FOR CHERRY SEED

134

DNA extraction from cherry seed

1. Use hammer to break hard seed coat. Take off papery coating around cotyledons

2. Split apart the cotyledons (not necessary, but makes it easier to crush)

3. Grind in liquid nitrogen. Try to prevent tissue from “jumping out” of the mortar when
crushing by covering the mortar with your hand

4. Quickly add 750ul of extraction buffer and transfer to 15m] orange cap tube. Add an
additional 7 SOul buffer to the mortar and then transfer it to the same 15ml tube.

Extraction Buffer

 

Final concentration Starting [] For 10 seed
200 mM Tris-HCl (pH 8.0) l M 3.6 ml
200 mM NaCl 5 M 720 ul
25 mM EDTA .5 M 900 ul
.5% SDS 20% 450 ul
H20 12.3 ml

5. Add 1.5ml of CTAB solution to each 15 ml tube

 

CTAB buffer solution

Final concentration Starting [] For 10 seed
2% CTAB 5% 7.2 ml
100 mM Tris-HCl (pH 8.0) 1 M 1.8 ml
20 mM EDTA .5 M 720 ul
1.4 M NaCl 5 M 5.04 ml
2% BME 360 ul
H20 2.88 ml

6. Invert a couple times and incubate 10 minutes at room temp
7. Add 3 ml chloroform2isoamyl alcohol (24:1).
8. Centrifuge 6500 rpm in for 10 min and transfer supernatant to new tubes
9. If any white chunks are in this supernatant after transferring it to the new tube, re-
centrifiige for an additional 10 minutes and transfer supernatant to new tube. Keep
repeating until there are no more white chunks.
10. Add 2/3 volume of isopropanol (about 1.6 mls) and incubate at room temp for 10 min
to precipitate DNA
11. Centrifuge at 6,500 rpm for 10 min. If there is no pellet, try re-centrifirging in the
tabletop centrifuge for a couple additional minutes. Remove supernatant. Careful not
to lose the little pellet

12. Wash DNA pellet with 70% ethanol, air dry, resuspend in about 40 ul TE, and add 1
ul RNase (10mg/ml)

135

APPENDIX B

COLLECTION OF SELF-INCOMPATIBILITY DATA AND SCHEMATIC
REPRESENTATIONS OF OB SERVED S-HAPLOTYPE SEGREGATION DATA

136

Table B.l: The self-pollination of 60 sour cherry selections to test the validity of the
hypothesis for the genetic control of SI and SC in sour cherry. Predictions

were based on the knowledge of the S-genotypes of each tree.

fi
I

 

Plant Maternal Paternal S-genotype SI/SC SI/SC
ID Parent ° Parent ° prediction phenotype

27 2(17) UF Surefire S13'SanS, SC SC
27 2(3 7) UP Surefire S13'SanSe SC SC
27 2(58) UF Surefire S13'SanSe SC SC
27 3(1) UF Surefire S13SanS, SC SC
27 3(24) UF Surefire S13'SanSe SC SC
27 3(42) UF Surefire S 1 3'SanSe SC SC
27 3(63) UF Surefire SquanS, SC SC
27 4(43) UF Surefire S13'SanSe SC SC
27 2(45) UF Surefire S4S;3vSaSe SC SC
27 3(16) UF Surefire S4S13vSaSe SC SC
27 3(65) UF Surefire 54513505, SC SC
27 2(61) UF Surefire S4Sz3'San SC SC
27 3(20) UF Surefire $45138an SC SC
27 2(23) UF Surefire S13'SaSeSx SC SC
27 2(32) UF Surefire S13-SaSeSx SC SC
27 2(43) UF Surefire S13vSaSan SC SC
27 3(46) UF Surefire S13'SaSeSx SC SC
27 3(48) UF Surefire S13'SanSx SC SC
27 4(10) UF Surefire S13vSanSx SC SC
27 2(24) UF Surefire S4S13'S0Sx SC SC
27 2(48) UF Surefire S4S13vSan SC SC
27 2(51) UF Surefire $45138an SC SC
27 2(57) UF Surefire 545136an SC SC
27 2(65) UF Surefire S4513'San SC SC
27 3(2) UF Surefire $5138an SC SC

137

(Continued)

 

Plant Maternal Paternal S-genotype SI/SC SI/SC
11) Parent ° Parent ° prediction phenotype
27 3(28) UF Surefire S4SI3'San SC SC
27 3(41) UF Surefire S4SI3'San SC SC
27 4(7) UF Surefire S4S13vSan SC SC
27 4(31) UF Surefire S4S13'San SC SC
27 13(32) UF RS Sl3'SaSeSx SC SC
27 13(51) UF RS S13'SdSeSx SC SC
27 13(59) UF RS S13’SdSeSx SC SC
27 13(65) UF RS S13'SdSeSx SC SC
27e 2(27) UF RS $136.65.. SC sc
27 13(45) UF RS S6SI3’SeSx SC SC
27 13(56) UF RS $65138an SC SC
27 13(57) UF RS 545135.251; SC SC
27e 2(28) UF RS S13vaSdSe SC SC
27 13(37) UF RS SzSaS'bSd SI SI
27 13(42) UF RS S4SoS'bSd SI SI
27 13(61) UF RS Sdasbsd SI SI
27 13(36) UF RS S4S6Sbe SI SI
27 9(12) UF [[1 18(12) S4SI3vSfo“ SC SC
27 9(14) UF III 18(12) Susasesx SC SC
27 9( 15) UP HI 18(12) SI3'SdSeSfa SC SC
27 9(23) UP 1111 18(12) S13'SdSeSfa SC SC
27 9(25) UP 1111 18(12) S4S13'Sde" SC SC
27 9(27) UP 111 18(12) S13’SdSeSfa SC SC
27 8(58) Mont Jubileum $5,585.." sc sc
27 8(59) Mont Jubileum Sagas,” so so
27 8(62) Mont Jubileum 51360925,, SC SC
27 12(51) UF Mont SanSeSx SC SC
27 23(19) UF Mont $13805de SC SC

138

(Continued)

 

Plant Maternal Paternal S-genotype SI/SC SI/SC
ID Parent Parent prediction phenotype
27 23(29) UF Mont SI3'Sa‘SeSx SC SC
27 23(35) UF Mont 513543an SC SC
27 12(54) UF Mont SI3"SdSeSx SC SI
27 12(50) UF Mont S4S6SoSe SC SC
27 23(22) UF Mont S4SxSI3'Sd SC SC
27 23(16) UF Mont S4S6San SI SI
27 23(42) UF Mont S4S6San SI SI

 

“ Sf represents a unique S-haplotype in III 18(12). The functionality of Sf is unknown.

b Sg represents a unique S-haplotype in Jubileum. The functionality of S8 is unknown.

° UF = Ujfehértoi fiirtos; RS = Rheinische Schattenmorelle; Mont = Montmorency

139

Table B.2: Thirteen sour cheny crosses used to test the validity of the hypothesis for the
genetic control of SI and SC in sour cherry. The compatibility or
incompatibility of each cross was predicted based on the S-genotype of the

parents.

 

Maternal Parent Paternal Parent Compatible (C) or Incompatible (I)

 

 

 

 

Plant 1]) S-genotype Plant ID S-genotype Prediction Phenotype
25 2(22) S4SsSbe 27 2(24) $45135an C C
27 2(24) SxSaSLSx 25 2(22) S4S6Sbe C C
25 2(22) S4S6Sbe 27 2(65) 543136an C C
27 2(65) $45135an 25 2(22) S4S6Sbe C C
25 3(35) S4S6Sasb 27 2(24) $45138an C C
27 2(24) $491365; 25 3(35) S4S6SaSb C C
25 3(35) S4SoS'aSb 27 2(65) S4S13vSan C C
27 2(65) S4Sr3'San 25 3(3 5) S4S6SaSb C C
25 3(4) 545135an 27 13(61) S4S6Sbe C C
27 13(61) $4565be 25 3(4) S13'SaS4Sx C C
27 13(61) 54.365be 25 3(28) $4565be I I
27 13(45) 56313'SeSx 25 3(28) $565be C C
27 13(57) S4SaSI3'Se 25 3(28) SbS4S6Sx C C

 

140

Table B.3: The S-genotype and SI or SC phenotype for 81 progeny in the Rheinische
Schattenmorelle (RS) x Erdi B6term6 (EB) population.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SI/SC SI/SC
Plant 11) S-genotype Prediction ‘ Phenotype b

25 2(7) S4S6Son SI or SC SI
25 2(15) 5456505.. SI or SC SC
25 2(23) 54363an SI or SC SI
25 2(36) S4S6San SI or SC SI
25 2(38) 54565an SI or SC -

25 2(44) S4S6San SI or SC SC
25 2(50) S4SaS'an SI or SC SI
25 2(52) $4553an SI or SC SC
25 2(62) S4S6S.S. 81 or SC SI
25 2(65) $4565an SI or SC SI
25 3(07) 54568an SI or SC -

25 3(21) SrsaSaS. SI or SC SC
25 3(34) S4S6San SI or SC SC
25 2(3) $45.5 ,. SI or SC SC
25 2(17) 54505be SI or SC SI
25 2(39) 565.5 ,. SC -

25 2(53) SaganSx SC SI °
25 3(29) S6S.S.Sx SC SC
25 2(33) SaSbeSx SC -

25 2(40) SaSbS,Sx SC -

25 2(28) SlySaSbe SC -

25 2(18) S4SI3'SaSb SC SC
25 2(32) StsntSaSb SC SC
25 2(46) $45,365, SC -

25 2(58) $45,319.51, SC SC
25 3(02) 55138.5, SC -

25 3(06) $45,555,, SC -

25 3(08) S4SI3'SaSb SC SC
25 2(16) 5,375.55. SC SC
25 2(8) 545135an SC SC
25 2(10) $45135an SC SC
25 2(12) $45,555,. SC SC

 

 

 

 

 

141

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SI/SC SI/SC
Plant ID S-genotype Prediction ‘ Phenotype b

25 2(14) 545135an SC -

25 2(20) $45135an SC -

25 2(25) S4S 1319an SC -

25 2(29) S45 1319an SC SC
25 2(3 5) 545135an SC SC
25 2(43) 545136an SC SC
25 2(47) S4SI3'Son SC SC
25 2(48) S4SI3'San SC SC
25 2(51) S4Sl3'San SC SC
25 2(55) $45135an SC SC
25 2(66) $451353an SC SC
25 3(04) $419138an SC SC
25 3(09) $45135an SC SC
25 3(14) $45135an SC SC
25 3(16) $45135an SC SC
25 3(31) $451319an SC SC
25 3(37) $451353an SC SC
25 2(05) 545651315}; SC SC
25 2(11) 5456513152; SC SC
25 2(49) 545651313}. SC SC
25 2(56) S4S6Sl3'Sa SC SC
25 2(63) 545651315}: SC SC
25 3(03) S4Sogz3'Sa SC SC
25 3(05) 545681315}: SC SC
25 3(20) 545551350 SC SC
25 2(30) S6SI3'San SC -

25 2(06) $456805}, SI SI
25 2(37) S4S6SaSb SI SI
25 2(42) 545650.31, SI SI
25 300) 54565051, SI -

25 3(32) S4S6SaSb SI SI
25 3(35) S4S6SaSb SI SI
25 2(13) S4S6Sbe SI SI
25 2(22) 54565be SI SI
25 2(41) 545(99be SI SI
25 2(45) $4565be SI SI

 

 

 

 

 

142

 

 

SI/SC
Plant 1]) S-genotype Prediction ‘ Phenotype b

SI/SC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

25 2(54) 54565be SI SI
25 2(60) 54565be SI SI
25 2(64) S4S§Sbe SI SI
25 3(25) S4SoSbe SI -
25 3(28) S4S6Sbe SI SI
25 2(2) 545135be SI -
25 2(59) S4SJ3'Sbe SI SI
25 3(13) 545135be SI SC 0
25 3(1 8) 5451319be SI -
25 3(22) $45135be SI SI
25 2(19) 545651355: SI SC c
25 2(27) $455135}: SI SI
25 3(24) 545651351: SI -

 

 

“ See Figure 3.1 for explanation of how the S1 or SC predictions were made
b The SI or SC phenotype was determined through observation of pollen tube grth
down self-pollinated styles. “-“ denotes individuals for which the SI or SC phenotype

could not be determined.

° The S1 or SC phenotypes of these three individuals were incorrectly predicted.

143

Chromosome pairing in RS

 

 

S6 Sa
S 13' Sb
Possible RS Gametes

Chromosome pairing in EB

 

 

Punnett Square

5., So
S 6m Sx
Possible EB Gametes

Note: Pollen containing S6," are
incompatible with RS styles

 

 

 

 

 

 

EB gametes
S483 S48)! Sasx
S6Sa S4S6SaSa S4S6San S6SoSan
sc sr sc
S6815 S4SoSaSb 5455be SoSbSan
SI SI SI
RS 313's. S4SI3'SaSa $45135an SI3'SaSan
gametes SC SC SC
Sl3’Sb 545135051; 545135be $135155an
sc sr so
56313' S4SoSJ3'Sa S4S6SI3’Sx S6SI3'Son
sc sr sc
Sash 54525me 545(15be SaSaSbe
so sr sc

 

 

 

 

 

Note: When progeny were S-genotyped, it was not possible to
determine dosage. Thus, S4S6SaSa appeared the same as S4S6San

Figure B.l: Graphic explanation of the expected S-genotypes and SI or SC phenotypes of
progeny in the Rheinische Schattenmorelle (RS) x Erdi B6term6 (EB)

population shown in Table B3.

144

Figure B.2: Schematic representation of the reciprocal inter-specific crosses between
sweet and sour cherry analyzed in Chapter 4. All possible pollen types are
shown on the stigma. Pollen tube growth either stops half-way down the
style (if the pollen tube encounters a cognate S-RNase that is functional) or
grows to the bottom of the pistil and fertilizes an egg (if the pollen tube does
not encounter its functional cognate S-RNase). The only S-RNases shown
in the styles are those that match an S-haplotype in the pollen. The non-
functional S6,,, and S6c-RNases from EB and Cigany 59, respectively, are
denoted by large X’s through the S-RNase. EF = Emperor Francis; UF =
Ujfehértoi fiirtos; EB = Erdi Boterrné; RS = Rheinische Schattenmorelle;

Mont = Montmorency.

145

Surefire (S4SJ3'San) x EF (S 3S4)

 

 

Concl: Surefire SrRNase is fimctional

UF ($290.95.) x Schmidt (5254)

$63

S4-RNase

 

Concl: UF SrRNase is functional

EB (S4S6mSan) X BF (S3S4)

 

 

Concl: EB SrRNase is functional

EF (S354) X Surefire (S4Sj3'San)

Concl: Surefire pollen-S4 gene is functional

EF (5354) X UF (S4Sa‘SeSx)

@@&@@®

S4-RNase

 

Concl: UF pollen-S4 gene is functional

EF (S 3S4) x EB (S4SgnSan)

WIS

Concl: EB pollen-S4 gene is functional

 

146

(Continued)
EB (3456,35,) x Gold (S356) Gold (S356) x EB (5456,55,)

are

S 6MXN386

 

 

 

Concl: EB San-RNase is not functional Concl: EB pollen-Sc... gene is functional

Cigany 59 (S6chSaSb) x Gold (S 3S6) Gold (S 3S6) x Cigany 59 (SngS'QSb)

cats

Sac-Xrlase

 

 

 

Concl: Cigany 59 Sac-RNase is not functional C0001: Cigany 59 pollen-Sac gene is functional

Cigany 59 (SachS'oSb) x Chelan (S 3S9)

 

 

Concl: Cigany 59 Sg-RNase is fimctional

147

RS (55136051,) X GOId (S 356)

$63

Sa-RNaS —.

 

Concl: RS So-RNase is functional

Mont ($68138an) X GOId (S 3S6)

 

 

Concl: Mont So-RNase is functional

(Continued)

GOld (S 355) X RS (S631 38051,)

W)
I

Concl: RS pollen-S6 gene is functional

GOId (S355) X Mont (19681350515)

 

 

Concl: Mont pollen-$6 gene is functional

148

Rheinische Schattenmorelle (SJ, 3.1931,)

Functionality of S-haplogpes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

S6 Functional Chromosome Pairing
$13: Non-Functional. Probably 36 Se
pollen-part mutant
Sa Non-Functional. Probably 813' 8"
pollen-part mutant
Sb Functional
Fragment sizes of S-RNases Gamete an”
S a S S
36 313' 8. Sb 56S S13 b .
Hindlll 5.8 kb 4.6 kb 6.4 kb 5.1 kb 65” 6513'
Xbal 5.5 kb 9.4 kb 2.4 kb 5 kb 31350 Sash
Pruc2lPCE 300 bp 620 bp 730 bp 550 bp . Requires multivalent
formation
Self-Pollination

a. I Imam

f

Sa-RNase?

Sa-RNase S13v-RNase .
Sb-RNase

 

Figure B.3: Summary of the S-haplotypes in Rheinische Schattenmorelle

149

Functionality of S-haplotxpes

Erdi BOtermo (Sagams'a)

S4 Functional

S6»:

in promoter

So

Non-Functional. S-RNase
mutant. 2600 bp insertion

Non-Functional. Probably

pollen-part mutant

Fragr_nent sizes of S-RNases

 

 

 

 

S4

86m

8.

 

Hindlll

5.6 +
6.1 kb

9kb

6.4 kb

 

Xbal

8.8 kb

5.5 kb

2.4 kb

 

 

Pruc2/PCE

 

 

850 bp 300 bp

 

730 bp

 

 

®c

Self-Pollination

£92®®

4%

\

S4-RNase

 

Figure B.4: Summary of the S-haplotypes in Erdi B6term6

150

 

W
84 8;
86m Sx
W

S4Sa S 6me
S4Sx S4S6m W
S6mSa San W

' Requires multivalent
formation

Sa-RNase?

Cigfiny 59 (S6cS9SaSb)

Functionality of S-haplogpes

S6c Non-Functional. S-RNase Q_—_ghromosome Pamn
mutant
866 S.

 

 

S9 Functional

 

 

Sa Non-Functional. Probably ' S9 Sb
pollen-part mutant

Sb Functional

 

 

 

 

 

 

 

 

 

 

 

Frament sizes of S-RNases w
S a S
86c $9 $3 Sb WWW WSb .
H d1“ 31 + :36ch S6cS9
in 5.8 kb ' 6.4 kb 5.1 kb ,
4.0 kb S980 SaSb
Xbal 5.5 kb 15 kb 2.4 kb 5 kb . . .
Requires multivalent
Prch/PCE 300 bp 550 bp 730 bp 550 bp formation
Self-Pollination

@1. I Iaaw

f-

Sb-RNase
Sg-RNase S.-RNase?

 

Figure B.5: Summary of the S-haplotypes in Cigany 59

151

Surefire (S4SI3Sa)

Functionality of S-haplogpes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. Chrom400some Pairing
S4 Functional
513' Non-Functional. Probably 8“ 8‘
pollen-part mutant
. 313' Sx
Sa Non-Functional. Probably
pollen-part mutant
Fragment sizes of S-RNases Gamete types
S a S 'Sx
S4 813. s. ‘S ’3 .
. 5 6 + S4Sx S4SI3’
Hindlll 6 1 kb 4.6 kb 6.4 kb ,
. SI3’Sa San
Xbal 8.8 kb 9.4 kb 2.4 kb . . .
Pruc2lPCE 850 bp 620 bp 730 bp If‘sr‘lggztf‘umvalent
Self-Pollination

@. I x®xfi®

?

S.—RNase?
S4-RNase 813' -RNase

 

Figure B.6: Summary of the S-haplotypes in Surefire

152

Montmorency (SoSJ13Sx)

Functionali of halo

 

 

 

 

So Functional Chromosome Pairing

513' Non-Functional. Probably 85 Sa
pollen-part mutant

5 - 313' Sx

Non-Functional. Probably
pollen-part mutant

 

 

 

 

Fragment sizes 9f S-RNases Gamete types
S a S 'Sx
86 813' Se 6S 13 *
Hindlll 5.8 kb 4.6 kb 6.4 kb 563* 56513'
Xbal 5.5 kb 9.4 kb 2.4 kb S136. Sasx '
Pruc2/PCE 300 bp 620 bp 730 bp - Requires multivalent

 

 

 

 

 

 

formation

Self-P llination

9a; 2951®A®®

Note: S. appears to be capable
of self-fertilization, but pollen
containing the pollen Sx-product

might be less competitive
Sa'RN as 3

Ss-RNase

S13o-RNase?

 

Figure B.7: Summary of the S-haplotypes in Montmorency

153

Ujfehértoi filrtlis (aspen)

Functionality of S-haplogpes

 

 

 

 

 

 

 

 

 

 

 

 

 

 
  

   
   

 

   

4 Functional Chromosome Pairing
Sd Non-Functional. Possibly S4 Sd
similar to Sa
8 8
Se Non-Functional. Pollen- e x
part mutant of S 1
Fragment sizes of S-RNases Gamete types
S S x
S4 3.. s. ‘S" ‘S .
S4Sx S4Se
Hindlll 5'6”” 6.2 kb 9.6 kb
6.1 kb S dSe S d5} "
Xbal 8.8 kb 2.4 kb ? kb . 1 a1
Requires mu tiv ent
Pruc2/PCE 850 bp 730 bp 620 bp formation
Self-Pollination
Note: S, appears to be capable
of self-fertilization, but pollen

     

Sd-RNas =
S4-RNase

Se-RNase?

 

containing the pollen Sx-product
may be less competitive

Figure B.8: Summary of the S-haplotypes in Ujfehértéi fiirtos

154

    

            

   

LIBRARIES

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